cancer immunotherapy combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is...

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The development of immunotherapeutics for oncology is based on the insight that the development of cancer, which involves progressive mutations, is monitored by the immune system. Most incipient tumours are elimi- nated by a process termed immunoediting or immune surveillance. The durable benefit produced by high-dose interleukin-2 (IL-2) or cytotoxic T lymphocyte antigen 4 (CTLA4) blockade is proof of principle that immuno- therapy can result in sustained antitumour responses. However, even before these therapeutics, there was clini- cal evidence of the immune system’s power to fight cancer (BOX 1). A subset of cancer mutations generate protein coding sequence changes called neoantigens, which can be processed into peptide antigens that are presented by the major histocompatibility complex (MHC) and recog- nized as foreign by T cells 1 . These neoantigens are the pri- mary targets for immunoediting 2,3 . Tumours that evade immunoediting often express molecules that inhibit the antitumour immune response, including programmed cell death 1 ligand 1 (PDL1; also known as B7-H1) 4 , indoleamine 2,3-dioxygenase (IDO), IL-10 and trans- forming growth factor-β (TGFβ). The immune response to cancer evolves over many years and ultimately fails; thus, the antitumour immune response has many charac- teristics of a chronic immune response, with T cell exhaus- tion mediated by the expression of multiple inhibitory receptors such as programmed cell death protein 1 (PD1), T cell immunoglobulin and mucin 3 (TIM3; also known as HAVCR2) and lymphocyte activation gene 3 protein (LAG3), among others 5 (FIG. 1). In this chronic response, interferon-γ (IFNγ) expressed by T cells upregulates PDL1 and IDO expression on tumour and infiltrating immune cells, forming a feedback loop that generates a PD1 signal that maintains immunosuppression in a dominant man- ner. This immunosuppressive tumour microenvironment is the major reason that many of the immunotherapies that aimed to directly stimulate the antitumour immune response failed. The discovery of the immunoinhibitory pathways that are upregulated in parallel with T lymphocyte acti- vation enabled a better understanding of the chronic inflammation process in infectious diseases and also uncovered powerful mechanisms by which tumours evade immune attack (FIGS 1,2; TABLE 1). These mech- anisms are frequently referred to as immune check- points or co-inhibitory pathways. The first-generation immune checkpoint inhibitor ipilimumab (Yervoy; Bristol-Myers Squibb), which gained US Food and Drug Administration (FDA) approval in 2011 (REF. 6), is a monoclonal antibody (mAb) that blocks CTLA4 and can induce sustained antitumour responses. We have learned much from clinical experience with ipilimumab (BOX 2). 1 Department of Medical Oncology, Dana–Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02215, USA. 2 Division of Haematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA. 3 SugarCone Biotech, Holliston, Massachusetts 01746, USA. 4 Videre Biotherapeutics, Watertown, Massachusetts 02472, USA. *These authors contributed equally to this work. Correspondence to G.J.F. e‑mail: gordon_freeman@ dfci.harvard.edu doi:10.1038/nrd4591 Combination cancer immunotherapy and new immunomodulatory targets Kathleen M. Mahoney 1,2 *, Paul D. Rennert 3,4 * and Gordon J. Freeman 1 Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory signals and enabling patients to produce an effective antitumour response. Inhibitors of CTLA4, PD1 or PDL1 administered as single agents have resulted in durable tumour regression in some patients, and combinations of PD1 and CTLA4 inhibitors may enhance antitumour benefit. Numerous additional immunomodulatory pathways as well as inhibitory factors expressed or secreted by myeloid and stromal cells in the tumour microenvironment are potential targets for synergizing with immune checkpoint blockade. Given the breadth of potential targets in the immune system, critical questions to address include which combinations should move forward in development and which patients will benefit from these treatments. This Review discusses the leading drug targets that are expressed on tumour cells and in the tumour microenvironment that allow enhancement of the antitumour immune response. CANCER IMMUNOTHERAPY REVIEWS NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 561 © 2015 Macmillan Publishers Limited. All rights reserved

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Page 1: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

The development of immunotherapeutics for oncology is based on the insight that the development of cancer which involves progressive mutations is monitored by the immune system Most incipient tumours are elimi-nated by a process termed immunoediting or immune surveillance The durable benefit produced by high-dose interleukin-2 (IL-2) or cytotoxic T lymphocyte antigen 4 (CTLA4) blockade is proof of principle that immuno-therapy can result in sustained antitumour responses However even before these therapeutics there was clini-cal evidence of the immune systemrsquos power to fight cancer (BOX 1) A subset of cancer mutations generate protein coding sequence changes called neoantigens which can be processed into peptide antigens that are presented by the major histocompatibility complex (MHC) and recog-nized as foreign by T cells1 These neoantigens are the pri-mary targets for immunoediting23 Tumours that evade immunoediting often express molecules that inhibit the antitumour immune response including programmed cell death 1 ligand 1 (PDL1 also known as B7-H1)4 indoleamine 23-dioxygenase (IDO) IL-10 and trans-forming growth factor-β (TGFβ) The immune response to cancer evolves over many years and ultimately fails thus the antitumour immune response has many charac-teristics of a chronic immune response with T cell exhaus-tion mediated by the expression of multiple inhibitory

receptors such as programmed cell death protein 1 (PD1) T cell immunoglobulin and mucin 3 (TIM3 also known as HAVCR2) and lymphocyte activation gene 3 protein (LAG3) among others5 (FIG 1) In this chronic response interferon-γ (IFNγ) expressed by T cells upregulates PDL1 and IDO expression on tumour and infiltrating immune cells forming a feedback loop that generates a PD1 signal that maintains immunosuppression in a dominant man-ner This immunosuppressive tumour microenvironment is the major reason that many of the immunotherapies that aimed to directly stimulate the antitumour immune response failed

The discovery of the immunoinhibitory pathways that are upregulated in parallel with T lymphocyte acti-vation enabled a better understanding of the chronic inflammation process in infectious diseases and also uncovered powerful mechanisms by which tumours evade immune attack (FIGS 12 TABLE 1) These mech-anisms are frequently referred to as immune check-points or co-inhibitory pathways The first-generation immune checkpoint inhibitor ipilimumab (Yervoy Bristol-Myers Squibb) which gained US Food and Drug Administration (FDA) approval in 2011 (REF 6) is a monoclonal antibody (mAb) that blocks CTLA4 and can induce sustained antitumour responses We have learned much from clinical experience with ipilimumab (BOX 2)

1Department of Medical Oncology DanandashFarber Cancer Institute Harvard Medical School Boston Massachusetts 02215 USA2Division of Haematology and Oncology Beth Israel Deaconess Medical Center Harvard Medical School Boston Massachusetts 02215 USA 3SugarCone Biotech Holliston Massachusetts 01746 USA 4Videre Biotherapeutics Watertown Massachusetts 02472 USA These authors contributed equally to this workCorrespondence to GJF e‑mail gordon_freemandfciharvardedudoi101038nrd4591

Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney12 Paul D Rennert34 and Gordon J Freeman1

Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory signals and enabling patients to produce an effective antitumour response Inhibitors of CTLA4 PD1 or PDL1 administered as single agents have resulted in durable tumour regression in some patients and combinations of PD1 and CTLA4 inhibitors may enhance antitumour benefit Numerous additional immunomodulatory pathways as well as inhibitory factors expressed or secreted by myeloid and stromal cells in the tumour microenvironment are potential targets for synergizing with immune checkpoint blockade Given the breadth of potential targets in the immune system critical questions to address include which combinations should move forward in development and which patients will benefit from these treatments This Review discusses the leading drug targets that are expressed on tumour cells and in the tumour microenvironment that allow enhancement of the antitumour immune response

C A N C E R I M M U N OT H E R A P Y

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 561

copy 2015 Macmillan Publishers Limited All rights reserved

ImmunoeditingImmunological processes that identify and eliminate cancer cells Also known as immune surveillance

Cytotoxic T lymphocyte antigen 4(CTLA4) An immune checkpoint that competes with CD28 for its B7 ligands on antigen presenting cells

NeoantigensNovel protein sequences that arise during cancer-associated mutagenesis and that can be processed into peptide antigens that are presented by MHC and recognized as foreign by T cells

Programmed cell death ligand 1(PDL1) An immune checkpoint ligand expressed on immune cells some normal tissues and many tumours PDL1 binds to programmed cell death protein 1 on lymphocytes to inhibit T cell receptor signalling and activation

T cell exhaustionA state of T cell non-responsiveness due to chronic activation

Programmed cell death protein 1(PD1) An immune checkpoint receptor expressed on activated lymphocytes and highly expressed on exhausted T lymphocytes

Interferon-γ(IFNγ) This cytokine triggers diverse cellular responses including macrophage activation and propagation of an immune response as well as the expression of negative regulatory factors such as programmed cell death 1 ligand 1 and indoleamine 23-dioxygenase

Immune checkpointsInhibitory pathways that regulate the adaptive immune responses

R-CHOP Acronym for the immunochemotherapy regimen used to treat non-Hodgkin lymphoma rituximab (R) cyclophosphamide (C) hydroxydaunorubicin (H) Oncovin (O Genus Pharmaceuticals vincristine) prednisone (P)

Combining anticancer therapies has been essential to achieve complete remission and cures for patients with cancer Immunochemotherapy the combination of immunotherapy and chemotherapy has become the standard of care for many tumours For example rituxi-mab (RituxanMabThera Roche) is a CD20-specific mAb that depletes CD20-expressing cells and thus has become a standard part of treatment for B cell lympho-mas for example R-CHOP However the immune check-point inhibitors function differently from most mAbs used in oncology (BOX 3) It is impressive that even as single agents checkpoint blockade can produce durable responses in some patients7ndash9

The next generation of immune checkpoint inhibitors block the interaction of PD1 on lymphocytes and PDL1 on antigen presenting cells (APCs) and tumour cells In the past 1ndash2 years multiple breakthrough designations for PD1- and PDL1-blocking mAbs have been granted by the FDA in melanoma Hodgkin lymphoma non-small-cell lung cancer (NSCLC) and bladder cancer Such designations have led to the accelerated FDA approval of pembrolizumab for patients with melanoma who are refractory to ipilimumab and of nivolumab for patients with melanoma or squamous cell NSCLC who are refrac-tory to ipilimumab Additional indications and therapeu-tics are expected to gain FDA approval in the near future Indeed the efficacy of immune checkpoint-blocking therapeutics in some of the more common cancers such as NSCLC which was previously considered not to be a candidate for immunotherapy is a landmark advance in cancer immunotherapy Additionally predictive bio-markers such as PDL1 status71011 illustrate how such assays may identify a subset of patients within a tumour type who may benefit more from single-agent immuno-therapy (BOX 4) For example a recent report indicated that mismatch repair deficiency is a potential biomarker for PD1 immunotherapy12 Response rates of up to 40 were achieved in mismatch repair-deficient tumours such as microsatellite instable colorectal cancer (n = 10) versus 0 in mismatch repair-proficient colorectal cancer

(n = 18)12 Mismatch repair-deficient tumours are associ-ated with a much higher neoantigen load than tumours with proficient mismatch repair prompting a Phase II trial investigating the potential role of immunotherapy in the treatment of tumours with mismatch repair deficits12

Two Phase III trials comparing ipilimumab with PD1 inhibitors (pembrolizumab (Keytruda Merck) or nivolumab (Opdivo Bristol-Myers Squibb)) in patients with melanoma found that PD1 blockade provided greater clinical benefit than ipilimumab1314 However time will tell whether the durable benefit seen with ipili-mumab which has produced responses lasting up to 10 years in some patients15 will be seen with PD1 blockers

Combination treatments with PD1 and CTLA4 inhib-itors have significantly increased objective response rates in melanoma1617 and are currently in Phase III trials in multiple tumour types Because immunoinhibition is dominant we believe that an immunotherapy regimen should begin with immune checkpoint blockade (for example a PD1 or CTLA4 antagonist) rather than a direct immune stimulator Release from immunoinhi-bition will open the door for combination with a large number of immunotherapies that directly stimulate the immune response Although combining PD1 and CTLA4 antagonists may improve response rates this particular combination appears to increase toxicity17 Novel combinations with an anti-PD1 pathway back-bone may produce better response rates in more tumour types with fewer side effects Candidates for combina-tions with immune checkpoint inhibitors are at various stages of clinical development (TABLE 1) These agents may directly stimulate cytotoxic T cells (FIG 1) block tumour-expressed immunoinhibitory factors (FIG 2) inhibit regulatory T (TReg) cells (FIG 3) block the inhi-bition of natural killer cell activity (FIG 4) or block the activity of soluble factors such as IDO produced by stromal myeloid and mesenchymal cells (FIG 5) Here we describe candidate targets for rational combination with PD1 pathway blockade many of which are currently in clinical development

Box 1 | Autoimmunity and the anticancer response by-product or bystander

Before the recent development of effective cancer immunotherapies there had been many hints of the potential for fighting cancer with the patientrsquos immune system For example patients with melanoma and vitiligo had a better prognosis than patients with melanoma who did not have vitiligo205206 Vitiligo is a result of an anti-melanocyte immune response essentially indicating an immune response against the tumour cell type The durable benefit produced by high-dose interleukin-2 (IL-2) is proof of principle that immunotherapy can result in sustained antitumour responses Benefit from treatment with IL-2 was associated with collateral autoimmunity including vitiligo207ndash209 Although only 26 of patients with melanoma who responded to IL-2 developed vitiligo no patients who had progressive disease developed vitiligo209 This phenomenon was first described in 1988 in patients receiving IL-2 and lymphokine-activated killer cells210 Early in the development of cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors immune-related adverse events were observed and associated with increased response rates6211212 In both melanoma and renal cell carcinoma the objective response rates with ipilimumab in patients with enterocolitis were 36 and 35 respectively versus 11 and 2 respectively in patients without enterocolitis213 However the role of autoimmunity as a surrogate end point in clinical practice is unclear particularly for immune checkpoint inhibitors Furthermore due to concern about immune-related adverse effects patients with autoimmune disease were largely excluded from immune checkpoint clinical trials Several recent case reports document that patients with autoimmune diseases such as rheumatoid arthritis and multiple sclerosis have tolerated checkpoint blockade generating an antitumour effect without exacerbation of autoimmune disease or development of other immune-related toxicity214 however no study has enrolled a significant number of patients with underlying autoimmunity

R E V I E W S

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Breakthrough designationsThe US Food and Drug Administration (FDA) designation for drugs that based on preliminary clinical evidence may substantially improve patient outcomes over available treatments Breakthrough designation gives access to intensive guidance from the FDA regarding product development

Objective response ratesPercentage of patients whose tumors decrease in size with therapy For example by the RECIST (Response Evaluation Criteria in Solid Tumours) criteria the percentage of patients whose tumours decrease by 30 or more

Figure 1 | T cell activation is a multiple-signal process The T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of naive T cells depends on a co-stimulatory signal through CD28 (signal 2) Additional interactions between ligands and activating (green shading) or inhibitory (red shading) receptors are crucial for further regulating T cell activation and tolerance These second signals include co-stimulatory ligands and receptors (blue) immune checkpoint ligands and receptors (red) tumour necrosis factor (TNF) receptor superfamily (TNFRSF brown) members and their trimeric TNF superfamily ligands (light brown) and additional members of the immunoglobulin superfamily (purple) Therapeutics targeting these and other pathways are in various stages of clinical development as indicated in TABLE 1 BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CTLA4 cytotoxic T lymphocyte antigen 4 GITR glucocorticoid-induced TNFR-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 PDL programmed cell death 1 ligand siglec sialic acid-binding immunoglobulin-type lectin TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 VISTA V-domain immunoglobulin suppressor of T cell activation

Nature Reviews | Drug Discovery

CD40

TL1A

GITR ligand

4-1BB ligand

OX40 ligand

CD70

HHLA2

ICOS ligand

CD86

CD80

MHC class II

VISTA

CD86

CD80

PDL1

PDL1

LAG3

CD40L

TNFRSF25

GITR

4-1BB

OX40

CD27

TMIGD2

ICOS

CD28

TCR

CTLA4

PD1

CD80

CD244

TIM3

BTLA

CD160

LIGHT

PDL2

BTNL2

B7-H3

B7-H4

Butyrophilin family

CD48

HVEM

Siglec family

Antigen presenting cell T cell

Galectin 9

Phosphatidylserine

Activation

Activation

Inhibition

Signal 1

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SuperagonistAn agent that produces a supra-physiological response greater than the response seen in normal physiology

Co-stimulatory signalA second non-antigen-specific signal that works with T cell receptor signalling to increase T cell activation

Checkpoint inhibition why combinationsA subset of patients with advanced cancers can respond to single-agent immune checkpoint blockade but most patients do not respond to such single-agent therapy Predictive biomarkers may provide a means to identify which patients will respond to monotherapy (BOX 4) Combining immunological agents may improve response rates and also improve the duration of response by stimulating an antitumour immunological memory Combinations of immunotherapies will require carefully planned Phase I dose-finding trials to assess the danger of overstimulating the immune system Such danger was illustrated by the clinical experience of targeting the T cell co-stimulatory receptor CD28 (REF 18) wherein 6 out of 6 subjects treated with a CD28 superagonist antibody developed life-threatening toxicity (a cytokine storm) in a Phase I trial leading to significant reluctance to further develop CD28 stimulatory agents19

Encouraging results from combinations with PD1 and PDL1 inhibitors have been reported from multi-ple Phase I trials including combinations with CTLA4 blockers with cytotoxic chemotherapy with radiation therapy or with small-molecule inhibitors such as the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors sunitinib (Sutent Pfizer) or pazopanib (Votrient Novartis)1620ndash26 Although toxici-ties appear to occur more frequently with such combina-tions the most significant toxicities are immune-related adverse effects which can be severe but largely manage-able with immunosuppressants Numerous clinical trials are underway to assess the safety and efficacy of novel

combinations However given the expense of Phase III trials careful development and rational design of combi-nation therapies during Phase III trials will be necessary to best incorporate the new therapeutics into current treatment algorithms and to determine the optimal timing and sequencing of regimens The underlying sci-entific rationale of each of the key therapeutics discussed herein can guide such rational design

Checkpoint blockade as a backboneThe rationale for combining CTLA4 and PD1 blockers is strong because although both CTLA4 and PD1 are expressed on T lymphocytes these pathways have dif-ferent mechanisms for inhibiting the function of these cells32728 CTLA4 competes with CD28 for CD80CD86 ligands and thereby blocks the CD28 co-stimulatory signal that is necessary for robust T cell activation and effector function By contrast PD1 is expressed on activated lym-phocytes and overexpressed on exhausted lymphocytes29 The interaction between PD1 and its ligands reduces T cell activation and decreases their cytotoxic activity

In a trial comparing the PD1-blocking mAb nivolumab to the alkylating agent dacarbazine in patients with previously untreated metastatic melanoma nivolumab elicited greater objective response rates (40 versus 139 P lt 0001) progression-free survival and overall survival30 A Phase I trial combining nivolumab and ipilimumab reported an objective response rate of 5316 and an even better response was observed in a randomized Phase II clinical trial comparing the com-bination versus ipilimumab alone (61 versus 11

Nature Reviews | Drug Discovery

CD30

CD70

HHLA2

ICOSL

MHC class I

PDL1

PDL2

B7-H3

B7-H4

Butyrophilin family

Galectin

Phosphatidylserine

Tumour cell Tumor Expression

Glioblastoma melanoma

Kidney prostate pancreatic glioblastoma

Breast renal cell ovarian oesophageal gastric pancreatic melanoma among others

Prostate renal cell non-small cell lung pancreatic gastric ovarian colorectal urothelial cell among others

Oesophageal ovarian pancreatic hepatocellular breast Hodgkin mediastinal large B-cell lymphoma among others

Melanoma renal cell head and neck cervical glioblastoma bladder oesophageal breast hepatocellular Hodgkin lymphoma mediastinallarge B-cell lymphoma among others

Non-Hodgkin lymphoma renal cell

Hodgkin lymphoma embryonal anaplastic large cell lymphoma

Non-small cell lung colorectal gastric among others

Breast lung thyroid melanoma pancreas ovary liver bladder colon prostate kidney oesophagus

Refs

189 196 223230-232

CD155

Ligands

PDL1

PDL2

B7-H3

B7-H4

HHLA2

Galectins

CD30

CD70

ICOSL

CD155

233ndash238

239ndash246

247ndash257

129

258ndash261

262

263 264

265 266

267

Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligands Individual tumours can express a selected subset of co-inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL inducible T cell co-stimulator ligand PDL programmed cell death 1 ligand

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Myeloid-derived suppressor cells(MDSCs) A heterogeneous population of myeloid lineage cells with immunosuppresive activities

Histone deactylaseAn intracellular protein that regulates gene transcription by modifying histones and thereby chromosome structure

BRAFA serinethreonine kinase often associated with solid tumour oncogenesis and resistance to therapy mutant BRAF may be an effective target for therapeutics as seen with vemurafenib in V600E-mutated melanoma

Cross-presentationThe ability of some antigen presenting cells to take up extracellular proteins or cells and present their antigens in the context of MHC class I

P lt 0001) in patients with previously untreated meta-static melanoma17 A Phase III trial comparing mono-therapy nivolumab and monotherapy ipilimumab to the combination in patients with melanoma found that nivolumab alone was less toxic and had greater clinical benefit than ipilimumab alone (ClinicalTrialsgov identi-fier NCT01844505 note that all trials cited in this arti-cle are from this database (see Further information)14 While the overall survival results are pending in this study nivolumab as monotherapy and in combination with ipilimumab had better objective response rates than ipilimumab alone (437 576 and 19 respectively) Moreover there is an ongoing clinical trial comparing the sequence of nivolumab and ipilimumab (NCT01783938) to further investigate the toxicities of these treatments The favourable efficacy and tolerability profile of the PD1 pathway-blocking agents has generated enthusi-asm for their combination with other FDA-approved antitumour agents including small-molecule targeting agents standard chemotherapy and radiation therapy31 Compared to CTLA4 the PD1 pathway has a more subtle role in maintaining peripheral tolerance and regulating inflammation32 Thus PD1ndashPDL1 blockade has taken the lead as the foundation of combination therapy in current clinical trials

Combinations with chemotherapy Historically immu-notherapy was considered to be most effective in cases in which there is a small burden of tumour this reason-ing also appears to be true for newer agents7 However accumulating evidence indicates that immunotherapies are effective in a broad range of tumours and therefore combining chemotherapy with immune checkpoint inhibitors may take advantage of three effects of chemo-therapy First to reduce the tumour burden second to potentiate the antitumour response by exposing neoanti-gen via necrosis of the tumour or third to directly affect the tumour stromal cells The choice of chemothera-peutic agent and timing of these combinations will be important because many cytotoxic chemotherapeutics target rapidly dividing cells Chemotherapy regimens that deplete proliferating lymphocytes may negatively affect the efficacy of therapeutics such as ipilimumab and nivolumab which act by facilitating the activa-tion and proliferation of tumour-infiltrating lympho-cytes Indeed increased absolute lymphocyte count may be a pharmacodynamic marker of the response to ipilimumab33

There is intriguing data to support the hypothesis that cytotoxic chemotherapy alters the immunosup-pressive microenvironment of the tumour Historically cyclophosphamide has been used to deplete TReg cells in preclinical adoptive T cell and vaccine models34 and this regimen may augment immunotherapies in patients Alternatively various chemotherapies such as 5-fluoro-uracil gemcitabine and taxanes can cause a decrease in myeloid-derived suppressor cells (MDSCs)3536 Some chemotherapy regimens may prime the immune sys-tem to better respond to immune checkpoint blockade Substantial data support the hypothesis that some chem-otherapeutic agents may function as a vaccine killing

tumour cells and increasing the amount of tumour antigen processed and presented to T cells (reviewed in REF 37) Chemotherapeutic agents that target epigenetic modifiers (for example histone deacetylase) are able to synergize with CTLA4 or PD1 blockers to eradicate pri-mary tumour and metastases in murine models of large established tumours that are poorly immunogenic38

Caution is warranted in developing combina-tion trials because the efficacy of standard therapy may be negatively affected Fortunately early reports of nivolumab in combination with standard-of-care platinum-based chemotherapy couplets (for exam-ple gemcitabinendashcisplatin pemetrexedndashcisplatin and paclitaxelndashcarboplatin) in a Phase I NSCLC trial have shown overall response rates similar to historical con-trols21 Discontinuation of therapy owing to treatment-related adverse effects was observed in 20 of patients Longer follow-up is necessary to determine whether the responses from these initial combination trials in NSCLC are more durable than standard chemotherapy Notably platinum-based chemotherapy causes sig-nificant nausea and taxanes can produce significant adverse reactions thus pretreatment with steroids is standard It is unclear how concurrent corticosteroid use will affect the efficacy of immune checkpoint inhibi-tors In mice corticosteroid administration abrogates the antitumour effect of lymphokine-activated killer cells and IL-2 (REF 39) In patients with advanced can-cer dexamethasone significantly reduced the toxicity of high-dose IL-2 but also significantly reduced the response rate to this potentially curative regimen4041 Although steroids are highly successful at reversing immune-related adverse effects due to immune check-point inhibitors the results with IL-2 and steroids raise concern that concurrent use will negatively affect the efficacy of other immunotherapies Although immune-related adverse effects are observed in patients treated with immune checkpoint blockers when steroids are used to treat such adverse effects they do not appear to abrogate the clinical benefit of treatment1417 Indeed a substantial number of patients in a study of ipilimumab plus nivolumab required immunosuppressive agents to treat toxicity (834) however of those who withdrew from the study owing to toxicity many responded to the combination (675 81 out of 120 patients)1442 A series of randomized clinical trials in NCSLC will be needed to determine the role of immune checkpoint blockade in combination or following the current standard therapy

Combinations with BRAF inhibitors With the renewed interest in immunotherapy the mechanism of action and resistance of many molecularly targeted agents is being re-examined and has been found to include immunological effects Treatment with the BRAF inhibi-tor vemurafenib (Zelboraf Roche) appears to improve the antitumour immune response to melanoma perhaps by increasing the cross-presentation of antigens from dead tumour cells4344 The development of resistance to BRAF inhibitors is accompanied by an increased expres-sion of PDL1 on the melanoma cells45 Studies using a mouse model of BRAFV600E mutant melanoma showed

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Table 1 | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

CTLA4 Ipilimumab FDA approved

Tremelimumab Phase III

PD1ndashPDL1 Pembrolizumab (PD1) FDA approved

Nivolumab (PD1) FDA approved

Atezolizumab (formerly MPDL3280A) (PDL1) Phase III

MEDI4736 (PDL1) Phase III

Avelumab (PDL1) Phase I

PDR001 (PD1) Phase I

TNF and TNFR superfamilies

4-1BBndash4-1BB ligand Urelumab PF-05082566 Phase II

OX40ndashOX40 ligand MEDI6469 Phase II

GITR TRX518 Phase I

CD27 Varlilumab Phase II

TNFRSF25ndashTL1A Preclinical

CD40ndashCD40 ligand CP-870893 Phase I

HVEMndashLIGHTndashLTA Preclinical

HVEMndashBTLAndashCD160 Preclinical

IgSF

LAG3 BMS-986016 Phase I

TIM3 Preclinical

Siglecs Preclinical

B7 and CD28‑related proteins

ICOSndashICOS ligand Preclinical

B7-H3 MGA271 Phase I

B7-H4 Preclinical

VISTA Preclinical

HHLA2ndashTMIGD2 Preclinical

Butyrophilins including BTNL2 Preclinical

CD244ndashCD48 Preclinical

TIGIT and PVR family members Preclinical

Natural killer cell targets

KIRs Lirilumab Phase II

ILTs and LIRs Preclinical

NKG2D and NKG2A IPH2201 Phase I

MICA and MICB Preclinical

CD244 Preclinical

Suppressive myeloid cells

CSF1R Emactuzumab Phase I

Soluble mediators

IDO INCB024360 Phase II

TGFβ Galunisertib Phase I

AdenosinendashCD39ndashCD73 Preclinical

CXCR4ndashCXCL12 Ulocuplumab BKT140 Phase III

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that PD1 or PDL1 blockade combined with BRAF inhibition increased the activity of tumour -infiltrating lymphocytes and prolonged survival46 Based on such observations clinical trials evaluating combinations of PD1 pathway inhibitors with BRAF inhibitors such as vemurafenib (NCT01656642) or dabrafenib (Tafinlar GlaxoSmithKline) and with MEK inhibi-tors such as trametinib (Mekinist GlaxoSmithKline) (NCT02224781) are now underway in melanoma Although a Phase I trial combining ipilimumab plus vemurafenib was terminated early owing to hepatotox-icity47 preliminary safety data from the combination trial of MEDI4736 (a PDL1-specific mAb developed by AstraZeneca) dabrafenib and trametinib appear promis-ing (n = 26) (NCT02027961)48

Combinations with VEGF-directed therapy The VEGFR tyrosine kinase inhibitor sunitinib blocks growth fac-tor signalling in tumour and vascular cells and also reduces the accumulation of MDSCs within tumours MDSCs are critical to the maintenance of the tumour microenvironment and are potent inhibitors of immune responses49 The impact of sunitinib on vascular cells and MDSCs is consistent with an impact on the tumour microenvironment a subject of substantial interest in rational immunotherapy design In studies of the effect of sunitinib on primary kidney cancer tumours 3 out of 8 kidney tumour specimens had low levels of MDSCs and normal T cell production of IFNγ whereas 5 out of 8 specimens showed high intratumoural MDSCs and low T cell production of IFNγ50 Such data have sup-ported the design of a Phase I trial combining nivolumab with either sunitinib or pazopanib as second-line ther-apy in patients with kidney cancer22 Notably response rates with the Phase I combinations were greater than expected for each agent individually22 Increased grade 3ndash4 toxicities observed with this combination were largely reversible by treatment with steroids or delaying or stopping further drug doses

Targeting VEGF directly may result in a more effec-tive combination than inhibiting the receptor Kidney cancers that progress on VEGFR tyrosine kinase inhibi-tor therapy are associated with increased VEGF produc-tion by the tumour51 Many tumour types also secrete VEGF and elevated VEGF serum levels are a marker of poor prognosis in diverse cancer indications52ndash54 VEGF can be immunosuppressive because its natural role is to support tissue remodelling and repair For example by removing signalling through VEGFR VEGF blockade can enhance dendritic cell function and subsequent T cell activation5556 Thus it is promising that the combination of the VEGF-specific mAb bevacizumab (Avastin GenentechRoche) and the PDL1-specific mAb atezolizumab (formerly known as MPDL3280A developed by GenentechRoche) has demonstrated only modest toxicity in Phase I trials57 Such observations pro-vide a rationale for why this combination therapy may be more effective in kidney cancer than monotherapy either as first-line therapy or after progression (when VEGF secretion is upregulated)

VEGF has many functions in addition to angio-genesis and can signal through receptors other than VEGF tyrosine kinase receptors For example neuro-pilins are multifunctional non-tyrosine kinase recep-tors that bind to specific isoforms of VEGF and to TGFβ and semaphorins58 Semaphorins are linked to tumour proliferation and survival in addition to their role in axonal guidance Neuropilin is also expressed on a subset of human TReg cells (FIG 3) and on a specialized subset of dendritic cells termed plasmacytoid dendritic cells although the functional relevance of this expres-sion in the immune system is unclear5960 Neuropilin is upregulated in multiple tumour types and expres-sion correlates with tumour progression61 A Phase I trial of MNRP1685A (a human immunoglobulin G1 (IgG1) mAb developed by Roche) which targets the VEGF-binding domain of neuropilin 1 demonstrated tolerability62 However a Phase Ib trial combining an

Other

Phosphatidylserine Bavituximab Phase IIIII

SIRPAndashCD47 CC-90002 Phase I

VEGF Bevacizumab FDA approved

Neuropilin MNRP1685A Phase I

BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CSF1R macrophage colony-stimulating factor receptor 1 CTLA4 cytotoxic T lymphocyte antigen 4 CXCL12 chemokine (C-X-C motif) ligand 12 CXCR4 C-X-C chemokine receptor type 4 GITR glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator IDO indoleamine 23-dioxygenase IgSF immunoglobulin superfamily ILT immunoglobulin-like transcript KIR killer inhibitory immunoglobulin-like receptor LAG3 lymphocyte activation gene 3 protein LIR leukocyte immunoglobulin-like LTA lymphotoxin-α MIC MHC class I polypeptide-related sequence PD1 programmed cell death protein 1 PDL1 programmed cell death 1 ligand 1 PVR poliovirus receptor SIRPA signal-regulatory protein- TGFβ transforming growth factor-β TIGIT T cell immunoreceptor with immunoglobulin and ITIM domains TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 TNFRSF25 TNFR superfamily member 25 TNFR TNF receptor VEGF vascular endothelial growth factor VISTA V-domain immunoglobulin suppressor of T cell activation Plerixafor (Mozobil GenzymeSanofi) is approved by the US Food and Drug Administration not as an antitumour therapy but as a bone marrow mobilizing agent for bone marrow transplantation

Table 1 (cont) | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

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TNF receptors(TNFRs) A large family of proteins associated with diverse cellular activities including the activation of lymphocytes

TNFR superfamily(TNFRSF) A group containing approximately 30 type I or type III membrane proteins and several secreted proteins TNFRSF receptors are characterized by the presence of one to four extracellular cysteine-rich ligand-binding domains

anti-neuropilin agent the VEGF-specific mAb beva-cizumab and chemotherapy resulted in greater than expected proteinuria63 This result suggests that dual blockade of both VEGF and neuropilin may be lim-ited by toxicity However the future development of a neuropilin-specific mAb may have a role independent of VEGF function by targeting the immune response through TReg cells The recent finding that semaphorin 4A signals through neuropilin 1 to regulate TReg cell function in the tumour environment suggests that tar-geting this pathway may deactivate TReg cells facilitat-ing the development of antitumour immunity64 (FIG 3) The role of neuropilin 1 on TReg cells is being intensively investigated with the hope that inhibiting this activity of neuropilin 1 can selectively target TReg cells

New targets the TNFR superfamilyConcurrent with the discovery and targeting of CD28 and CTLA4 members of the tumour necrosis factor (TNF) family of proteins were identified and developed as some of the first molecularly defined immunothera-peutics Local infusion of TNF and lymphotoxin-α has

been used as a treatment for primary melanoma and sarcoma for decades although with limited efficacy and pronounced toxicity65 Other early attempts targeted various TNF receptors (TNFRs) with agonist antibodies to directly induce tumour cell death Examples of this class of therapeutics include antibodies directed against TRAIL receptor 2 (also known as TNFRSF10B) lymphotoxin-β receptor (also known as TNFRSF3) and FN14 (also known as TNFRSF12A) Some of these approaches were limited by toxicity notably hepatic toxicity The next wave of antibody development target-ing this receptor family came with the identification of 4-1BB (also known as CD137TNFRSF9) and related TNFRs as critical regulators of previously activated T cells66 There are six receptors in the TNFR superfamily (TNFRSF) currently receiving attention as immune-activating agents these are discussed below In contrast to immune checkpoint targets such as PD1 the goal of most of these antibodies is to activate the receptors thus they act as agonists (FIG 1) The driving concept for com-bination therapy is that TNFRSF agonists will further activate and support the antitumour immune response released by immune checkpoint inhibition

4-1BB 4-1BB is a co-stimulatory receptor on T cells and natural killer cells and it is expressed on neutrophils and myeloid lineage cells including some subpopula-tions of dendritic cells Ligation of 4-1BB on T cells and natural killer cells induces cell activation survival and enhances effector functions 4-1BB is expressed rapidly after T cell activation and its expression continues for up to 48 hours 4-1BB is found on all activated T cell subsets including TReg cells The pleiotropic activity of 4-1BB agonist antibodies is apparent in preclinical studies in which the effects varied depending on the models Preclinical data demonstrated 4-1BB effects on CD8+ T cell proliferation and survival In some model systems effects on CD4+ T cells have been reported The role of 4-1BB in TReg cell function is confusing as conflicting data suggest that 4-1BB activity can either expand or restrict TReg cell activation Importantly 41bb knockout mice can develop autoimmunity suggesting a role for 4-1BB in maintaining immune homeostasis67 Furthermore 41bb knockout mice are deficient in their ability to reject tumours in tumour challenge mod-els demonstrating that effective antitumour memory requires 4-1BB Some 4-1BB-deficient mice spontane-ously develop B cell lymphomas as they age but whether this is a consequence of underlying proliferation of auto-immune B cells or defective immunoediting is unclear

Two agonistic 4-1BB-specific antibodies urelumab (an IgG4 mAb developed by Bristol-Meyers Squibb) and PF-05082566 (an IgG2 mAb developed by Pfizer) are in Phase I trials Preliminary findings support the hypoth-esis that 4-1BB-specific agonist activity will increase T cell and natural killer cell proliferation and activity68 A Phase II trial of urelumab in patients with melanoma was terminated early owing to a high incidence of grade 4 hepatotoxicity69 New trials with urelumab are under-way to establish a safe and efficacious dose includ-ing a monotherapy trial in patients with advanced or

Box 2 | Lessons learnt from CTLA4 inhibitors

In both preclinical and clinical models cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors were developed in combination with other anticancer therapies Anti-CTLA4 monotherapy was not effective in the poorly immunogenic B16 melanoma mouse model however the combination of a CTLA4 inhibitor with a granulocytendashmacrophage colony stimulating factor (GM-CSF)-producing tumour vaccine resulted in tumour rejection as well as immune-related vitiligo215 Surviving mice also rejected tumour re-challenge supporting the hypothesis that CTLA4 blockade can support the development of antitumour immune memory In clinical trials the combination of ipilimumab with the gp100 peptide vaccine did not improve the clinical benefit of ipilimumab6 Moreover in a Phase II trial of high-dose ipilimumab (10 mg per kg) and GM-CSF greater antitumour responses were observed and with less toxicity than with ipilimumab alone216 It is unclear whether GM-CSF is playing a protective role that limits toxicity Preclinical studies have shown that GM-CSF can recruit dendritic cells but also enhance regulatory T (T

Reg) cells

providing possible mechanisms for enhanced activity and reduced toxicity217 These results successfully informed the design of a Phase III trial

It is clear that development of anti-CTLA4 combination therapies have been limited by toxicity as seen in Phase I trials that combine CTLA4 inhibitors with small-molecule agents For example adding ipilimumab after 1 month of treatment with the BRAF inhibitor vemurafenib caused undue toxicity Eight of the first 10 patients enrolled developed asymptomatic grade 3 liver toxicity within 5 weeks of starting ipilimumab which was reversible with drug discontinuation or active suppression of the immune response with glucocorticoid steroids47 Similarly the dose-limiting toxicity observed in the combination trial of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor sunitinib with the CTLA4-blocking antibody tremelimumab was rapid-onset acute kidney injury that was reversible with steroids218 Both trials were terminated due to toxicity Separate from its toxicity profile mechanistically anti-CTLA4 therapy may not be an optimal backbone for improving efficacy with other therapies When ipilimumab was combined with high-dose IL-2 ipilimumab failed to show any synergy producing only a 22 response rate little more than either therapy alone219 Similarly the Phase III trial combining ipilimumab with the chemotherapy dacarbazine demonstrated that ipilimumab in combination with dacarbazine provided an improved survival benefit over dacarbazine alone but the survival rate was comparable with subsequent trials evaluating ipilimumab as monotherapy6220 Although some combinations may fail to synergize the antitumour effect others may even blunt the antitumour effect Therefore given the multitude of potential combinations rational design is essential

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Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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copy 2015 Macmillan Publishers Limited All rights reserved

antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 2: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

ImmunoeditingImmunological processes that identify and eliminate cancer cells Also known as immune surveillance

Cytotoxic T lymphocyte antigen 4(CTLA4) An immune checkpoint that competes with CD28 for its B7 ligands on antigen presenting cells

NeoantigensNovel protein sequences that arise during cancer-associated mutagenesis and that can be processed into peptide antigens that are presented by MHC and recognized as foreign by T cells

Programmed cell death ligand 1(PDL1) An immune checkpoint ligand expressed on immune cells some normal tissues and many tumours PDL1 binds to programmed cell death protein 1 on lymphocytes to inhibit T cell receptor signalling and activation

T cell exhaustionA state of T cell non-responsiveness due to chronic activation

Programmed cell death protein 1(PD1) An immune checkpoint receptor expressed on activated lymphocytes and highly expressed on exhausted T lymphocytes

Interferon-γ(IFNγ) This cytokine triggers diverse cellular responses including macrophage activation and propagation of an immune response as well as the expression of negative regulatory factors such as programmed cell death 1 ligand 1 and indoleamine 23-dioxygenase

Immune checkpointsInhibitory pathways that regulate the adaptive immune responses

R-CHOP Acronym for the immunochemotherapy regimen used to treat non-Hodgkin lymphoma rituximab (R) cyclophosphamide (C) hydroxydaunorubicin (H) Oncovin (O Genus Pharmaceuticals vincristine) prednisone (P)

Combining anticancer therapies has been essential to achieve complete remission and cures for patients with cancer Immunochemotherapy the combination of immunotherapy and chemotherapy has become the standard of care for many tumours For example rituxi-mab (RituxanMabThera Roche) is a CD20-specific mAb that depletes CD20-expressing cells and thus has become a standard part of treatment for B cell lympho-mas for example R-CHOP However the immune check-point inhibitors function differently from most mAbs used in oncology (BOX 3) It is impressive that even as single agents checkpoint blockade can produce durable responses in some patients7ndash9

The next generation of immune checkpoint inhibitors block the interaction of PD1 on lymphocytes and PDL1 on antigen presenting cells (APCs) and tumour cells In the past 1ndash2 years multiple breakthrough designations for PD1- and PDL1-blocking mAbs have been granted by the FDA in melanoma Hodgkin lymphoma non-small-cell lung cancer (NSCLC) and bladder cancer Such designations have led to the accelerated FDA approval of pembrolizumab for patients with melanoma who are refractory to ipilimumab and of nivolumab for patients with melanoma or squamous cell NSCLC who are refrac-tory to ipilimumab Additional indications and therapeu-tics are expected to gain FDA approval in the near future Indeed the efficacy of immune checkpoint-blocking therapeutics in some of the more common cancers such as NSCLC which was previously considered not to be a candidate for immunotherapy is a landmark advance in cancer immunotherapy Additionally predictive bio-markers such as PDL1 status71011 illustrate how such assays may identify a subset of patients within a tumour type who may benefit more from single-agent immuno-therapy (BOX 4) For example a recent report indicated that mismatch repair deficiency is a potential biomarker for PD1 immunotherapy12 Response rates of up to 40 were achieved in mismatch repair-deficient tumours such as microsatellite instable colorectal cancer (n = 10) versus 0 in mismatch repair-proficient colorectal cancer

(n = 18)12 Mismatch repair-deficient tumours are associ-ated with a much higher neoantigen load than tumours with proficient mismatch repair prompting a Phase II trial investigating the potential role of immunotherapy in the treatment of tumours with mismatch repair deficits12

Two Phase III trials comparing ipilimumab with PD1 inhibitors (pembrolizumab (Keytruda Merck) or nivolumab (Opdivo Bristol-Myers Squibb)) in patients with melanoma found that PD1 blockade provided greater clinical benefit than ipilimumab1314 However time will tell whether the durable benefit seen with ipili-mumab which has produced responses lasting up to 10 years in some patients15 will be seen with PD1 blockers

Combination treatments with PD1 and CTLA4 inhib-itors have significantly increased objective response rates in melanoma1617 and are currently in Phase III trials in multiple tumour types Because immunoinhibition is dominant we believe that an immunotherapy regimen should begin with immune checkpoint blockade (for example a PD1 or CTLA4 antagonist) rather than a direct immune stimulator Release from immunoinhi-bition will open the door for combination with a large number of immunotherapies that directly stimulate the immune response Although combining PD1 and CTLA4 antagonists may improve response rates this particular combination appears to increase toxicity17 Novel combinations with an anti-PD1 pathway back-bone may produce better response rates in more tumour types with fewer side effects Candidates for combina-tions with immune checkpoint inhibitors are at various stages of clinical development (TABLE 1) These agents may directly stimulate cytotoxic T cells (FIG 1) block tumour-expressed immunoinhibitory factors (FIG 2) inhibit regulatory T (TReg) cells (FIG 3) block the inhi-bition of natural killer cell activity (FIG 4) or block the activity of soluble factors such as IDO produced by stromal myeloid and mesenchymal cells (FIG 5) Here we describe candidate targets for rational combination with PD1 pathway blockade many of which are currently in clinical development

Box 1 | Autoimmunity and the anticancer response by-product or bystander

Before the recent development of effective cancer immunotherapies there had been many hints of the potential for fighting cancer with the patientrsquos immune system For example patients with melanoma and vitiligo had a better prognosis than patients with melanoma who did not have vitiligo205206 Vitiligo is a result of an anti-melanocyte immune response essentially indicating an immune response against the tumour cell type The durable benefit produced by high-dose interleukin-2 (IL-2) is proof of principle that immunotherapy can result in sustained antitumour responses Benefit from treatment with IL-2 was associated with collateral autoimmunity including vitiligo207ndash209 Although only 26 of patients with melanoma who responded to IL-2 developed vitiligo no patients who had progressive disease developed vitiligo209 This phenomenon was first described in 1988 in patients receiving IL-2 and lymphokine-activated killer cells210 Early in the development of cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors immune-related adverse events were observed and associated with increased response rates6211212 In both melanoma and renal cell carcinoma the objective response rates with ipilimumab in patients with enterocolitis were 36 and 35 respectively versus 11 and 2 respectively in patients without enterocolitis213 However the role of autoimmunity as a surrogate end point in clinical practice is unclear particularly for immune checkpoint inhibitors Furthermore due to concern about immune-related adverse effects patients with autoimmune disease were largely excluded from immune checkpoint clinical trials Several recent case reports document that patients with autoimmune diseases such as rheumatoid arthritis and multiple sclerosis have tolerated checkpoint blockade generating an antitumour effect without exacerbation of autoimmune disease or development of other immune-related toxicity214 however no study has enrolled a significant number of patients with underlying autoimmunity

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Breakthrough designationsThe US Food and Drug Administration (FDA) designation for drugs that based on preliminary clinical evidence may substantially improve patient outcomes over available treatments Breakthrough designation gives access to intensive guidance from the FDA regarding product development

Objective response ratesPercentage of patients whose tumors decrease in size with therapy For example by the RECIST (Response Evaluation Criteria in Solid Tumours) criteria the percentage of patients whose tumours decrease by 30 or more

Figure 1 | T cell activation is a multiple-signal process The T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of naive T cells depends on a co-stimulatory signal through CD28 (signal 2) Additional interactions between ligands and activating (green shading) or inhibitory (red shading) receptors are crucial for further regulating T cell activation and tolerance These second signals include co-stimulatory ligands and receptors (blue) immune checkpoint ligands and receptors (red) tumour necrosis factor (TNF) receptor superfamily (TNFRSF brown) members and their trimeric TNF superfamily ligands (light brown) and additional members of the immunoglobulin superfamily (purple) Therapeutics targeting these and other pathways are in various stages of clinical development as indicated in TABLE 1 BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CTLA4 cytotoxic T lymphocyte antigen 4 GITR glucocorticoid-induced TNFR-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 PDL programmed cell death 1 ligand siglec sialic acid-binding immunoglobulin-type lectin TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 VISTA V-domain immunoglobulin suppressor of T cell activation

Nature Reviews | Drug Discovery

CD40

TL1A

GITR ligand

4-1BB ligand

OX40 ligand

CD70

HHLA2

ICOS ligand

CD86

CD80

MHC class II

VISTA

CD86

CD80

PDL1

PDL1

LAG3

CD40L

TNFRSF25

GITR

4-1BB

OX40

CD27

TMIGD2

ICOS

CD28

TCR

CTLA4

PD1

CD80

CD244

TIM3

BTLA

CD160

LIGHT

PDL2

BTNL2

B7-H3

B7-H4

Butyrophilin family

CD48

HVEM

Siglec family

Antigen presenting cell T cell

Galectin 9

Phosphatidylserine

Activation

Activation

Inhibition

Signal 1

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SuperagonistAn agent that produces a supra-physiological response greater than the response seen in normal physiology

Co-stimulatory signalA second non-antigen-specific signal that works with T cell receptor signalling to increase T cell activation

Checkpoint inhibition why combinationsA subset of patients with advanced cancers can respond to single-agent immune checkpoint blockade but most patients do not respond to such single-agent therapy Predictive biomarkers may provide a means to identify which patients will respond to monotherapy (BOX 4) Combining immunological agents may improve response rates and also improve the duration of response by stimulating an antitumour immunological memory Combinations of immunotherapies will require carefully planned Phase I dose-finding trials to assess the danger of overstimulating the immune system Such danger was illustrated by the clinical experience of targeting the T cell co-stimulatory receptor CD28 (REF 18) wherein 6 out of 6 subjects treated with a CD28 superagonist antibody developed life-threatening toxicity (a cytokine storm) in a Phase I trial leading to significant reluctance to further develop CD28 stimulatory agents19

Encouraging results from combinations with PD1 and PDL1 inhibitors have been reported from multi-ple Phase I trials including combinations with CTLA4 blockers with cytotoxic chemotherapy with radiation therapy or with small-molecule inhibitors such as the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors sunitinib (Sutent Pfizer) or pazopanib (Votrient Novartis)1620ndash26 Although toxici-ties appear to occur more frequently with such combina-tions the most significant toxicities are immune-related adverse effects which can be severe but largely manage-able with immunosuppressants Numerous clinical trials are underway to assess the safety and efficacy of novel

combinations However given the expense of Phase III trials careful development and rational design of combi-nation therapies during Phase III trials will be necessary to best incorporate the new therapeutics into current treatment algorithms and to determine the optimal timing and sequencing of regimens The underlying sci-entific rationale of each of the key therapeutics discussed herein can guide such rational design

Checkpoint blockade as a backboneThe rationale for combining CTLA4 and PD1 blockers is strong because although both CTLA4 and PD1 are expressed on T lymphocytes these pathways have dif-ferent mechanisms for inhibiting the function of these cells32728 CTLA4 competes with CD28 for CD80CD86 ligands and thereby blocks the CD28 co-stimulatory signal that is necessary for robust T cell activation and effector function By contrast PD1 is expressed on activated lym-phocytes and overexpressed on exhausted lymphocytes29 The interaction between PD1 and its ligands reduces T cell activation and decreases their cytotoxic activity

In a trial comparing the PD1-blocking mAb nivolumab to the alkylating agent dacarbazine in patients with previously untreated metastatic melanoma nivolumab elicited greater objective response rates (40 versus 139 P lt 0001) progression-free survival and overall survival30 A Phase I trial combining nivolumab and ipilimumab reported an objective response rate of 5316 and an even better response was observed in a randomized Phase II clinical trial comparing the com-bination versus ipilimumab alone (61 versus 11

Nature Reviews | Drug Discovery

CD30

CD70

HHLA2

ICOSL

MHC class I

PDL1

PDL2

B7-H3

B7-H4

Butyrophilin family

Galectin

Phosphatidylserine

Tumour cell Tumor Expression

Glioblastoma melanoma

Kidney prostate pancreatic glioblastoma

Breast renal cell ovarian oesophageal gastric pancreatic melanoma among others

Prostate renal cell non-small cell lung pancreatic gastric ovarian colorectal urothelial cell among others

Oesophageal ovarian pancreatic hepatocellular breast Hodgkin mediastinal large B-cell lymphoma among others

Melanoma renal cell head and neck cervical glioblastoma bladder oesophageal breast hepatocellular Hodgkin lymphoma mediastinallarge B-cell lymphoma among others

Non-Hodgkin lymphoma renal cell

Hodgkin lymphoma embryonal anaplastic large cell lymphoma

Non-small cell lung colorectal gastric among others

Breast lung thyroid melanoma pancreas ovary liver bladder colon prostate kidney oesophagus

Refs

189 196 223230-232

CD155

Ligands

PDL1

PDL2

B7-H3

B7-H4

HHLA2

Galectins

CD30

CD70

ICOSL

CD155

233ndash238

239ndash246

247ndash257

129

258ndash261

262

263 264

265 266

267

Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligands Individual tumours can express a selected subset of co-inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL inducible T cell co-stimulator ligand PDL programmed cell death 1 ligand

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Myeloid-derived suppressor cells(MDSCs) A heterogeneous population of myeloid lineage cells with immunosuppresive activities

Histone deactylaseAn intracellular protein that regulates gene transcription by modifying histones and thereby chromosome structure

BRAFA serinethreonine kinase often associated with solid tumour oncogenesis and resistance to therapy mutant BRAF may be an effective target for therapeutics as seen with vemurafenib in V600E-mutated melanoma

Cross-presentationThe ability of some antigen presenting cells to take up extracellular proteins or cells and present their antigens in the context of MHC class I

P lt 0001) in patients with previously untreated meta-static melanoma17 A Phase III trial comparing mono-therapy nivolumab and monotherapy ipilimumab to the combination in patients with melanoma found that nivolumab alone was less toxic and had greater clinical benefit than ipilimumab alone (ClinicalTrialsgov identi-fier NCT01844505 note that all trials cited in this arti-cle are from this database (see Further information)14 While the overall survival results are pending in this study nivolumab as monotherapy and in combination with ipilimumab had better objective response rates than ipilimumab alone (437 576 and 19 respectively) Moreover there is an ongoing clinical trial comparing the sequence of nivolumab and ipilimumab (NCT01783938) to further investigate the toxicities of these treatments The favourable efficacy and tolerability profile of the PD1 pathway-blocking agents has generated enthusi-asm for their combination with other FDA-approved antitumour agents including small-molecule targeting agents standard chemotherapy and radiation therapy31 Compared to CTLA4 the PD1 pathway has a more subtle role in maintaining peripheral tolerance and regulating inflammation32 Thus PD1ndashPDL1 blockade has taken the lead as the foundation of combination therapy in current clinical trials

Combinations with chemotherapy Historically immu-notherapy was considered to be most effective in cases in which there is a small burden of tumour this reason-ing also appears to be true for newer agents7 However accumulating evidence indicates that immunotherapies are effective in a broad range of tumours and therefore combining chemotherapy with immune checkpoint inhibitors may take advantage of three effects of chemo-therapy First to reduce the tumour burden second to potentiate the antitumour response by exposing neoanti-gen via necrosis of the tumour or third to directly affect the tumour stromal cells The choice of chemothera-peutic agent and timing of these combinations will be important because many cytotoxic chemotherapeutics target rapidly dividing cells Chemotherapy regimens that deplete proliferating lymphocytes may negatively affect the efficacy of therapeutics such as ipilimumab and nivolumab which act by facilitating the activa-tion and proliferation of tumour-infiltrating lympho-cytes Indeed increased absolute lymphocyte count may be a pharmacodynamic marker of the response to ipilimumab33

There is intriguing data to support the hypothesis that cytotoxic chemotherapy alters the immunosup-pressive microenvironment of the tumour Historically cyclophosphamide has been used to deplete TReg cells in preclinical adoptive T cell and vaccine models34 and this regimen may augment immunotherapies in patients Alternatively various chemotherapies such as 5-fluoro-uracil gemcitabine and taxanes can cause a decrease in myeloid-derived suppressor cells (MDSCs)3536 Some chemotherapy regimens may prime the immune sys-tem to better respond to immune checkpoint blockade Substantial data support the hypothesis that some chem-otherapeutic agents may function as a vaccine killing

tumour cells and increasing the amount of tumour antigen processed and presented to T cells (reviewed in REF 37) Chemotherapeutic agents that target epigenetic modifiers (for example histone deacetylase) are able to synergize with CTLA4 or PD1 blockers to eradicate pri-mary tumour and metastases in murine models of large established tumours that are poorly immunogenic38

Caution is warranted in developing combina-tion trials because the efficacy of standard therapy may be negatively affected Fortunately early reports of nivolumab in combination with standard-of-care platinum-based chemotherapy couplets (for exam-ple gemcitabinendashcisplatin pemetrexedndashcisplatin and paclitaxelndashcarboplatin) in a Phase I NSCLC trial have shown overall response rates similar to historical con-trols21 Discontinuation of therapy owing to treatment-related adverse effects was observed in 20 of patients Longer follow-up is necessary to determine whether the responses from these initial combination trials in NSCLC are more durable than standard chemotherapy Notably platinum-based chemotherapy causes sig-nificant nausea and taxanes can produce significant adverse reactions thus pretreatment with steroids is standard It is unclear how concurrent corticosteroid use will affect the efficacy of immune checkpoint inhibi-tors In mice corticosteroid administration abrogates the antitumour effect of lymphokine-activated killer cells and IL-2 (REF 39) In patients with advanced can-cer dexamethasone significantly reduced the toxicity of high-dose IL-2 but also significantly reduced the response rate to this potentially curative regimen4041 Although steroids are highly successful at reversing immune-related adverse effects due to immune check-point inhibitors the results with IL-2 and steroids raise concern that concurrent use will negatively affect the efficacy of other immunotherapies Although immune-related adverse effects are observed in patients treated with immune checkpoint blockers when steroids are used to treat such adverse effects they do not appear to abrogate the clinical benefit of treatment1417 Indeed a substantial number of patients in a study of ipilimumab plus nivolumab required immunosuppressive agents to treat toxicity (834) however of those who withdrew from the study owing to toxicity many responded to the combination (675 81 out of 120 patients)1442 A series of randomized clinical trials in NCSLC will be needed to determine the role of immune checkpoint blockade in combination or following the current standard therapy

Combinations with BRAF inhibitors With the renewed interest in immunotherapy the mechanism of action and resistance of many molecularly targeted agents is being re-examined and has been found to include immunological effects Treatment with the BRAF inhibi-tor vemurafenib (Zelboraf Roche) appears to improve the antitumour immune response to melanoma perhaps by increasing the cross-presentation of antigens from dead tumour cells4344 The development of resistance to BRAF inhibitors is accompanied by an increased expres-sion of PDL1 on the melanoma cells45 Studies using a mouse model of BRAFV600E mutant melanoma showed

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Table 1 | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

CTLA4 Ipilimumab FDA approved

Tremelimumab Phase III

PD1ndashPDL1 Pembrolizumab (PD1) FDA approved

Nivolumab (PD1) FDA approved

Atezolizumab (formerly MPDL3280A) (PDL1) Phase III

MEDI4736 (PDL1) Phase III

Avelumab (PDL1) Phase I

PDR001 (PD1) Phase I

TNF and TNFR superfamilies

4-1BBndash4-1BB ligand Urelumab PF-05082566 Phase II

OX40ndashOX40 ligand MEDI6469 Phase II

GITR TRX518 Phase I

CD27 Varlilumab Phase II

TNFRSF25ndashTL1A Preclinical

CD40ndashCD40 ligand CP-870893 Phase I

HVEMndashLIGHTndashLTA Preclinical

HVEMndashBTLAndashCD160 Preclinical

IgSF

LAG3 BMS-986016 Phase I

TIM3 Preclinical

Siglecs Preclinical

B7 and CD28‑related proteins

ICOSndashICOS ligand Preclinical

B7-H3 MGA271 Phase I

B7-H4 Preclinical

VISTA Preclinical

HHLA2ndashTMIGD2 Preclinical

Butyrophilins including BTNL2 Preclinical

CD244ndashCD48 Preclinical

TIGIT and PVR family members Preclinical

Natural killer cell targets

KIRs Lirilumab Phase II

ILTs and LIRs Preclinical

NKG2D and NKG2A IPH2201 Phase I

MICA and MICB Preclinical

CD244 Preclinical

Suppressive myeloid cells

CSF1R Emactuzumab Phase I

Soluble mediators

IDO INCB024360 Phase II

TGFβ Galunisertib Phase I

AdenosinendashCD39ndashCD73 Preclinical

CXCR4ndashCXCL12 Ulocuplumab BKT140 Phase III

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that PD1 or PDL1 blockade combined with BRAF inhibition increased the activity of tumour -infiltrating lymphocytes and prolonged survival46 Based on such observations clinical trials evaluating combinations of PD1 pathway inhibitors with BRAF inhibitors such as vemurafenib (NCT01656642) or dabrafenib (Tafinlar GlaxoSmithKline) and with MEK inhibi-tors such as trametinib (Mekinist GlaxoSmithKline) (NCT02224781) are now underway in melanoma Although a Phase I trial combining ipilimumab plus vemurafenib was terminated early owing to hepatotox-icity47 preliminary safety data from the combination trial of MEDI4736 (a PDL1-specific mAb developed by AstraZeneca) dabrafenib and trametinib appear promis-ing (n = 26) (NCT02027961)48

Combinations with VEGF-directed therapy The VEGFR tyrosine kinase inhibitor sunitinib blocks growth fac-tor signalling in tumour and vascular cells and also reduces the accumulation of MDSCs within tumours MDSCs are critical to the maintenance of the tumour microenvironment and are potent inhibitors of immune responses49 The impact of sunitinib on vascular cells and MDSCs is consistent with an impact on the tumour microenvironment a subject of substantial interest in rational immunotherapy design In studies of the effect of sunitinib on primary kidney cancer tumours 3 out of 8 kidney tumour specimens had low levels of MDSCs and normal T cell production of IFNγ whereas 5 out of 8 specimens showed high intratumoural MDSCs and low T cell production of IFNγ50 Such data have sup-ported the design of a Phase I trial combining nivolumab with either sunitinib or pazopanib as second-line ther-apy in patients with kidney cancer22 Notably response rates with the Phase I combinations were greater than expected for each agent individually22 Increased grade 3ndash4 toxicities observed with this combination were largely reversible by treatment with steroids or delaying or stopping further drug doses

Targeting VEGF directly may result in a more effec-tive combination than inhibiting the receptor Kidney cancers that progress on VEGFR tyrosine kinase inhibi-tor therapy are associated with increased VEGF produc-tion by the tumour51 Many tumour types also secrete VEGF and elevated VEGF serum levels are a marker of poor prognosis in diverse cancer indications52ndash54 VEGF can be immunosuppressive because its natural role is to support tissue remodelling and repair For example by removing signalling through VEGFR VEGF blockade can enhance dendritic cell function and subsequent T cell activation5556 Thus it is promising that the combination of the VEGF-specific mAb bevacizumab (Avastin GenentechRoche) and the PDL1-specific mAb atezolizumab (formerly known as MPDL3280A developed by GenentechRoche) has demonstrated only modest toxicity in Phase I trials57 Such observations pro-vide a rationale for why this combination therapy may be more effective in kidney cancer than monotherapy either as first-line therapy or after progression (when VEGF secretion is upregulated)

VEGF has many functions in addition to angio-genesis and can signal through receptors other than VEGF tyrosine kinase receptors For example neuro-pilins are multifunctional non-tyrosine kinase recep-tors that bind to specific isoforms of VEGF and to TGFβ and semaphorins58 Semaphorins are linked to tumour proliferation and survival in addition to their role in axonal guidance Neuropilin is also expressed on a subset of human TReg cells (FIG 3) and on a specialized subset of dendritic cells termed plasmacytoid dendritic cells although the functional relevance of this expres-sion in the immune system is unclear5960 Neuropilin is upregulated in multiple tumour types and expres-sion correlates with tumour progression61 A Phase I trial of MNRP1685A (a human immunoglobulin G1 (IgG1) mAb developed by Roche) which targets the VEGF-binding domain of neuropilin 1 demonstrated tolerability62 However a Phase Ib trial combining an

Other

Phosphatidylserine Bavituximab Phase IIIII

SIRPAndashCD47 CC-90002 Phase I

VEGF Bevacizumab FDA approved

Neuropilin MNRP1685A Phase I

BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CSF1R macrophage colony-stimulating factor receptor 1 CTLA4 cytotoxic T lymphocyte antigen 4 CXCL12 chemokine (C-X-C motif) ligand 12 CXCR4 C-X-C chemokine receptor type 4 GITR glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator IDO indoleamine 23-dioxygenase IgSF immunoglobulin superfamily ILT immunoglobulin-like transcript KIR killer inhibitory immunoglobulin-like receptor LAG3 lymphocyte activation gene 3 protein LIR leukocyte immunoglobulin-like LTA lymphotoxin-α MIC MHC class I polypeptide-related sequence PD1 programmed cell death protein 1 PDL1 programmed cell death 1 ligand 1 PVR poliovirus receptor SIRPA signal-regulatory protein- TGFβ transforming growth factor-β TIGIT T cell immunoreceptor with immunoglobulin and ITIM domains TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 TNFRSF25 TNFR superfamily member 25 TNFR TNF receptor VEGF vascular endothelial growth factor VISTA V-domain immunoglobulin suppressor of T cell activation Plerixafor (Mozobil GenzymeSanofi) is approved by the US Food and Drug Administration not as an antitumour therapy but as a bone marrow mobilizing agent for bone marrow transplantation

Table 1 (cont) | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

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TNF receptors(TNFRs) A large family of proteins associated with diverse cellular activities including the activation of lymphocytes

TNFR superfamily(TNFRSF) A group containing approximately 30 type I or type III membrane proteins and several secreted proteins TNFRSF receptors are characterized by the presence of one to four extracellular cysteine-rich ligand-binding domains

anti-neuropilin agent the VEGF-specific mAb beva-cizumab and chemotherapy resulted in greater than expected proteinuria63 This result suggests that dual blockade of both VEGF and neuropilin may be lim-ited by toxicity However the future development of a neuropilin-specific mAb may have a role independent of VEGF function by targeting the immune response through TReg cells The recent finding that semaphorin 4A signals through neuropilin 1 to regulate TReg cell function in the tumour environment suggests that tar-geting this pathway may deactivate TReg cells facilitat-ing the development of antitumour immunity64 (FIG 3) The role of neuropilin 1 on TReg cells is being intensively investigated with the hope that inhibiting this activity of neuropilin 1 can selectively target TReg cells

New targets the TNFR superfamilyConcurrent with the discovery and targeting of CD28 and CTLA4 members of the tumour necrosis factor (TNF) family of proteins were identified and developed as some of the first molecularly defined immunothera-peutics Local infusion of TNF and lymphotoxin-α has

been used as a treatment for primary melanoma and sarcoma for decades although with limited efficacy and pronounced toxicity65 Other early attempts targeted various TNF receptors (TNFRs) with agonist antibodies to directly induce tumour cell death Examples of this class of therapeutics include antibodies directed against TRAIL receptor 2 (also known as TNFRSF10B) lymphotoxin-β receptor (also known as TNFRSF3) and FN14 (also known as TNFRSF12A) Some of these approaches were limited by toxicity notably hepatic toxicity The next wave of antibody development target-ing this receptor family came with the identification of 4-1BB (also known as CD137TNFRSF9) and related TNFRs as critical regulators of previously activated T cells66 There are six receptors in the TNFR superfamily (TNFRSF) currently receiving attention as immune-activating agents these are discussed below In contrast to immune checkpoint targets such as PD1 the goal of most of these antibodies is to activate the receptors thus they act as agonists (FIG 1) The driving concept for com-bination therapy is that TNFRSF agonists will further activate and support the antitumour immune response released by immune checkpoint inhibition

4-1BB 4-1BB is a co-stimulatory receptor on T cells and natural killer cells and it is expressed on neutrophils and myeloid lineage cells including some subpopula-tions of dendritic cells Ligation of 4-1BB on T cells and natural killer cells induces cell activation survival and enhances effector functions 4-1BB is expressed rapidly after T cell activation and its expression continues for up to 48 hours 4-1BB is found on all activated T cell subsets including TReg cells The pleiotropic activity of 4-1BB agonist antibodies is apparent in preclinical studies in which the effects varied depending on the models Preclinical data demonstrated 4-1BB effects on CD8+ T cell proliferation and survival In some model systems effects on CD4+ T cells have been reported The role of 4-1BB in TReg cell function is confusing as conflicting data suggest that 4-1BB activity can either expand or restrict TReg cell activation Importantly 41bb knockout mice can develop autoimmunity suggesting a role for 4-1BB in maintaining immune homeostasis67 Furthermore 41bb knockout mice are deficient in their ability to reject tumours in tumour challenge mod-els demonstrating that effective antitumour memory requires 4-1BB Some 4-1BB-deficient mice spontane-ously develop B cell lymphomas as they age but whether this is a consequence of underlying proliferation of auto-immune B cells or defective immunoediting is unclear

Two agonistic 4-1BB-specific antibodies urelumab (an IgG4 mAb developed by Bristol-Meyers Squibb) and PF-05082566 (an IgG2 mAb developed by Pfizer) are in Phase I trials Preliminary findings support the hypoth-esis that 4-1BB-specific agonist activity will increase T cell and natural killer cell proliferation and activity68 A Phase II trial of urelumab in patients with melanoma was terminated early owing to a high incidence of grade 4 hepatotoxicity69 New trials with urelumab are under-way to establish a safe and efficacious dose includ-ing a monotherapy trial in patients with advanced or

Box 2 | Lessons learnt from CTLA4 inhibitors

In both preclinical and clinical models cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors were developed in combination with other anticancer therapies Anti-CTLA4 monotherapy was not effective in the poorly immunogenic B16 melanoma mouse model however the combination of a CTLA4 inhibitor with a granulocytendashmacrophage colony stimulating factor (GM-CSF)-producing tumour vaccine resulted in tumour rejection as well as immune-related vitiligo215 Surviving mice also rejected tumour re-challenge supporting the hypothesis that CTLA4 blockade can support the development of antitumour immune memory In clinical trials the combination of ipilimumab with the gp100 peptide vaccine did not improve the clinical benefit of ipilimumab6 Moreover in a Phase II trial of high-dose ipilimumab (10 mg per kg) and GM-CSF greater antitumour responses were observed and with less toxicity than with ipilimumab alone216 It is unclear whether GM-CSF is playing a protective role that limits toxicity Preclinical studies have shown that GM-CSF can recruit dendritic cells but also enhance regulatory T (T

Reg) cells

providing possible mechanisms for enhanced activity and reduced toxicity217 These results successfully informed the design of a Phase III trial

It is clear that development of anti-CTLA4 combination therapies have been limited by toxicity as seen in Phase I trials that combine CTLA4 inhibitors with small-molecule agents For example adding ipilimumab after 1 month of treatment with the BRAF inhibitor vemurafenib caused undue toxicity Eight of the first 10 patients enrolled developed asymptomatic grade 3 liver toxicity within 5 weeks of starting ipilimumab which was reversible with drug discontinuation or active suppression of the immune response with glucocorticoid steroids47 Similarly the dose-limiting toxicity observed in the combination trial of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor sunitinib with the CTLA4-blocking antibody tremelimumab was rapid-onset acute kidney injury that was reversible with steroids218 Both trials were terminated due to toxicity Separate from its toxicity profile mechanistically anti-CTLA4 therapy may not be an optimal backbone for improving efficacy with other therapies When ipilimumab was combined with high-dose IL-2 ipilimumab failed to show any synergy producing only a 22 response rate little more than either therapy alone219 Similarly the Phase III trial combining ipilimumab with the chemotherapy dacarbazine demonstrated that ipilimumab in combination with dacarbazine provided an improved survival benefit over dacarbazine alone but the survival rate was comparable with subsequent trials evaluating ipilimumab as monotherapy6220 Although some combinations may fail to synergize the antitumour effect others may even blunt the antitumour effect Therefore given the multitude of potential combinations rational design is essential

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Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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copy 2015 Macmillan Publishers Limited All rights reserved

antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 3: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

Breakthrough designationsThe US Food and Drug Administration (FDA) designation for drugs that based on preliminary clinical evidence may substantially improve patient outcomes over available treatments Breakthrough designation gives access to intensive guidance from the FDA regarding product development

Objective response ratesPercentage of patients whose tumors decrease in size with therapy For example by the RECIST (Response Evaluation Criteria in Solid Tumours) criteria the percentage of patients whose tumours decrease by 30 or more

Figure 1 | T cell activation is a multiple-signal process The T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of naive T cells depends on a co-stimulatory signal through CD28 (signal 2) Additional interactions between ligands and activating (green shading) or inhibitory (red shading) receptors are crucial for further regulating T cell activation and tolerance These second signals include co-stimulatory ligands and receptors (blue) immune checkpoint ligands and receptors (red) tumour necrosis factor (TNF) receptor superfamily (TNFRSF brown) members and their trimeric TNF superfamily ligands (light brown) and additional members of the immunoglobulin superfamily (purple) Therapeutics targeting these and other pathways are in various stages of clinical development as indicated in TABLE 1 BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CTLA4 cytotoxic T lymphocyte antigen 4 GITR glucocorticoid-induced TNFR-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 PDL programmed cell death 1 ligand siglec sialic acid-binding immunoglobulin-type lectin TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 VISTA V-domain immunoglobulin suppressor of T cell activation

Nature Reviews | Drug Discovery

CD40

TL1A

GITR ligand

4-1BB ligand

OX40 ligand

CD70

HHLA2

ICOS ligand

CD86

CD80

MHC class II

VISTA

CD86

CD80

PDL1

PDL1

LAG3

CD40L

TNFRSF25

GITR

4-1BB

OX40

CD27

TMIGD2

ICOS

CD28

TCR

CTLA4

PD1

CD80

CD244

TIM3

BTLA

CD160

LIGHT

PDL2

BTNL2

B7-H3

B7-H4

Butyrophilin family

CD48

HVEM

Siglec family

Antigen presenting cell T cell

Galectin 9

Phosphatidylserine

Activation

Activation

Inhibition

Signal 1

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SuperagonistAn agent that produces a supra-physiological response greater than the response seen in normal physiology

Co-stimulatory signalA second non-antigen-specific signal that works with T cell receptor signalling to increase T cell activation

Checkpoint inhibition why combinationsA subset of patients with advanced cancers can respond to single-agent immune checkpoint blockade but most patients do not respond to such single-agent therapy Predictive biomarkers may provide a means to identify which patients will respond to monotherapy (BOX 4) Combining immunological agents may improve response rates and also improve the duration of response by stimulating an antitumour immunological memory Combinations of immunotherapies will require carefully planned Phase I dose-finding trials to assess the danger of overstimulating the immune system Such danger was illustrated by the clinical experience of targeting the T cell co-stimulatory receptor CD28 (REF 18) wherein 6 out of 6 subjects treated with a CD28 superagonist antibody developed life-threatening toxicity (a cytokine storm) in a Phase I trial leading to significant reluctance to further develop CD28 stimulatory agents19

Encouraging results from combinations with PD1 and PDL1 inhibitors have been reported from multi-ple Phase I trials including combinations with CTLA4 blockers with cytotoxic chemotherapy with radiation therapy or with small-molecule inhibitors such as the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors sunitinib (Sutent Pfizer) or pazopanib (Votrient Novartis)1620ndash26 Although toxici-ties appear to occur more frequently with such combina-tions the most significant toxicities are immune-related adverse effects which can be severe but largely manage-able with immunosuppressants Numerous clinical trials are underway to assess the safety and efficacy of novel

combinations However given the expense of Phase III trials careful development and rational design of combi-nation therapies during Phase III trials will be necessary to best incorporate the new therapeutics into current treatment algorithms and to determine the optimal timing and sequencing of regimens The underlying sci-entific rationale of each of the key therapeutics discussed herein can guide such rational design

Checkpoint blockade as a backboneThe rationale for combining CTLA4 and PD1 blockers is strong because although both CTLA4 and PD1 are expressed on T lymphocytes these pathways have dif-ferent mechanisms for inhibiting the function of these cells32728 CTLA4 competes with CD28 for CD80CD86 ligands and thereby blocks the CD28 co-stimulatory signal that is necessary for robust T cell activation and effector function By contrast PD1 is expressed on activated lym-phocytes and overexpressed on exhausted lymphocytes29 The interaction between PD1 and its ligands reduces T cell activation and decreases their cytotoxic activity

In a trial comparing the PD1-blocking mAb nivolumab to the alkylating agent dacarbazine in patients with previously untreated metastatic melanoma nivolumab elicited greater objective response rates (40 versus 139 P lt 0001) progression-free survival and overall survival30 A Phase I trial combining nivolumab and ipilimumab reported an objective response rate of 5316 and an even better response was observed in a randomized Phase II clinical trial comparing the com-bination versus ipilimumab alone (61 versus 11

Nature Reviews | Drug Discovery

CD30

CD70

HHLA2

ICOSL

MHC class I

PDL1

PDL2

B7-H3

B7-H4

Butyrophilin family

Galectin

Phosphatidylserine

Tumour cell Tumor Expression

Glioblastoma melanoma

Kidney prostate pancreatic glioblastoma

Breast renal cell ovarian oesophageal gastric pancreatic melanoma among others

Prostate renal cell non-small cell lung pancreatic gastric ovarian colorectal urothelial cell among others

Oesophageal ovarian pancreatic hepatocellular breast Hodgkin mediastinal large B-cell lymphoma among others

Melanoma renal cell head and neck cervical glioblastoma bladder oesophageal breast hepatocellular Hodgkin lymphoma mediastinallarge B-cell lymphoma among others

Non-Hodgkin lymphoma renal cell

Hodgkin lymphoma embryonal anaplastic large cell lymphoma

Non-small cell lung colorectal gastric among others

Breast lung thyroid melanoma pancreas ovary liver bladder colon prostate kidney oesophagus

Refs

189 196 223230-232

CD155

Ligands

PDL1

PDL2

B7-H3

B7-H4

HHLA2

Galectins

CD30

CD70

ICOSL

CD155

233ndash238

239ndash246

247ndash257

129

258ndash261

262

263 264

265 266

267

Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligands Individual tumours can express a selected subset of co-inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL inducible T cell co-stimulator ligand PDL programmed cell death 1 ligand

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Myeloid-derived suppressor cells(MDSCs) A heterogeneous population of myeloid lineage cells with immunosuppresive activities

Histone deactylaseAn intracellular protein that regulates gene transcription by modifying histones and thereby chromosome structure

BRAFA serinethreonine kinase often associated with solid tumour oncogenesis and resistance to therapy mutant BRAF may be an effective target for therapeutics as seen with vemurafenib in V600E-mutated melanoma

Cross-presentationThe ability of some antigen presenting cells to take up extracellular proteins or cells and present their antigens in the context of MHC class I

P lt 0001) in patients with previously untreated meta-static melanoma17 A Phase III trial comparing mono-therapy nivolumab and monotherapy ipilimumab to the combination in patients with melanoma found that nivolumab alone was less toxic and had greater clinical benefit than ipilimumab alone (ClinicalTrialsgov identi-fier NCT01844505 note that all trials cited in this arti-cle are from this database (see Further information)14 While the overall survival results are pending in this study nivolumab as monotherapy and in combination with ipilimumab had better objective response rates than ipilimumab alone (437 576 and 19 respectively) Moreover there is an ongoing clinical trial comparing the sequence of nivolumab and ipilimumab (NCT01783938) to further investigate the toxicities of these treatments The favourable efficacy and tolerability profile of the PD1 pathway-blocking agents has generated enthusi-asm for their combination with other FDA-approved antitumour agents including small-molecule targeting agents standard chemotherapy and radiation therapy31 Compared to CTLA4 the PD1 pathway has a more subtle role in maintaining peripheral tolerance and regulating inflammation32 Thus PD1ndashPDL1 blockade has taken the lead as the foundation of combination therapy in current clinical trials

Combinations with chemotherapy Historically immu-notherapy was considered to be most effective in cases in which there is a small burden of tumour this reason-ing also appears to be true for newer agents7 However accumulating evidence indicates that immunotherapies are effective in a broad range of tumours and therefore combining chemotherapy with immune checkpoint inhibitors may take advantage of three effects of chemo-therapy First to reduce the tumour burden second to potentiate the antitumour response by exposing neoanti-gen via necrosis of the tumour or third to directly affect the tumour stromal cells The choice of chemothera-peutic agent and timing of these combinations will be important because many cytotoxic chemotherapeutics target rapidly dividing cells Chemotherapy regimens that deplete proliferating lymphocytes may negatively affect the efficacy of therapeutics such as ipilimumab and nivolumab which act by facilitating the activa-tion and proliferation of tumour-infiltrating lympho-cytes Indeed increased absolute lymphocyte count may be a pharmacodynamic marker of the response to ipilimumab33

There is intriguing data to support the hypothesis that cytotoxic chemotherapy alters the immunosup-pressive microenvironment of the tumour Historically cyclophosphamide has been used to deplete TReg cells in preclinical adoptive T cell and vaccine models34 and this regimen may augment immunotherapies in patients Alternatively various chemotherapies such as 5-fluoro-uracil gemcitabine and taxanes can cause a decrease in myeloid-derived suppressor cells (MDSCs)3536 Some chemotherapy regimens may prime the immune sys-tem to better respond to immune checkpoint blockade Substantial data support the hypothesis that some chem-otherapeutic agents may function as a vaccine killing

tumour cells and increasing the amount of tumour antigen processed and presented to T cells (reviewed in REF 37) Chemotherapeutic agents that target epigenetic modifiers (for example histone deacetylase) are able to synergize with CTLA4 or PD1 blockers to eradicate pri-mary tumour and metastases in murine models of large established tumours that are poorly immunogenic38

Caution is warranted in developing combina-tion trials because the efficacy of standard therapy may be negatively affected Fortunately early reports of nivolumab in combination with standard-of-care platinum-based chemotherapy couplets (for exam-ple gemcitabinendashcisplatin pemetrexedndashcisplatin and paclitaxelndashcarboplatin) in a Phase I NSCLC trial have shown overall response rates similar to historical con-trols21 Discontinuation of therapy owing to treatment-related adverse effects was observed in 20 of patients Longer follow-up is necessary to determine whether the responses from these initial combination trials in NSCLC are more durable than standard chemotherapy Notably platinum-based chemotherapy causes sig-nificant nausea and taxanes can produce significant adverse reactions thus pretreatment with steroids is standard It is unclear how concurrent corticosteroid use will affect the efficacy of immune checkpoint inhibi-tors In mice corticosteroid administration abrogates the antitumour effect of lymphokine-activated killer cells and IL-2 (REF 39) In patients with advanced can-cer dexamethasone significantly reduced the toxicity of high-dose IL-2 but also significantly reduced the response rate to this potentially curative regimen4041 Although steroids are highly successful at reversing immune-related adverse effects due to immune check-point inhibitors the results with IL-2 and steroids raise concern that concurrent use will negatively affect the efficacy of other immunotherapies Although immune-related adverse effects are observed in patients treated with immune checkpoint blockers when steroids are used to treat such adverse effects they do not appear to abrogate the clinical benefit of treatment1417 Indeed a substantial number of patients in a study of ipilimumab plus nivolumab required immunosuppressive agents to treat toxicity (834) however of those who withdrew from the study owing to toxicity many responded to the combination (675 81 out of 120 patients)1442 A series of randomized clinical trials in NCSLC will be needed to determine the role of immune checkpoint blockade in combination or following the current standard therapy

Combinations with BRAF inhibitors With the renewed interest in immunotherapy the mechanism of action and resistance of many molecularly targeted agents is being re-examined and has been found to include immunological effects Treatment with the BRAF inhibi-tor vemurafenib (Zelboraf Roche) appears to improve the antitumour immune response to melanoma perhaps by increasing the cross-presentation of antigens from dead tumour cells4344 The development of resistance to BRAF inhibitors is accompanied by an increased expres-sion of PDL1 on the melanoma cells45 Studies using a mouse model of BRAFV600E mutant melanoma showed

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Table 1 | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

CTLA4 Ipilimumab FDA approved

Tremelimumab Phase III

PD1ndashPDL1 Pembrolizumab (PD1) FDA approved

Nivolumab (PD1) FDA approved

Atezolizumab (formerly MPDL3280A) (PDL1) Phase III

MEDI4736 (PDL1) Phase III

Avelumab (PDL1) Phase I

PDR001 (PD1) Phase I

TNF and TNFR superfamilies

4-1BBndash4-1BB ligand Urelumab PF-05082566 Phase II

OX40ndashOX40 ligand MEDI6469 Phase II

GITR TRX518 Phase I

CD27 Varlilumab Phase II

TNFRSF25ndashTL1A Preclinical

CD40ndashCD40 ligand CP-870893 Phase I

HVEMndashLIGHTndashLTA Preclinical

HVEMndashBTLAndashCD160 Preclinical

IgSF

LAG3 BMS-986016 Phase I

TIM3 Preclinical

Siglecs Preclinical

B7 and CD28‑related proteins

ICOSndashICOS ligand Preclinical

B7-H3 MGA271 Phase I

B7-H4 Preclinical

VISTA Preclinical

HHLA2ndashTMIGD2 Preclinical

Butyrophilins including BTNL2 Preclinical

CD244ndashCD48 Preclinical

TIGIT and PVR family members Preclinical

Natural killer cell targets

KIRs Lirilumab Phase II

ILTs and LIRs Preclinical

NKG2D and NKG2A IPH2201 Phase I

MICA and MICB Preclinical

CD244 Preclinical

Suppressive myeloid cells

CSF1R Emactuzumab Phase I

Soluble mediators

IDO INCB024360 Phase II

TGFβ Galunisertib Phase I

AdenosinendashCD39ndashCD73 Preclinical

CXCR4ndashCXCL12 Ulocuplumab BKT140 Phase III

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that PD1 or PDL1 blockade combined with BRAF inhibition increased the activity of tumour -infiltrating lymphocytes and prolonged survival46 Based on such observations clinical trials evaluating combinations of PD1 pathway inhibitors with BRAF inhibitors such as vemurafenib (NCT01656642) or dabrafenib (Tafinlar GlaxoSmithKline) and with MEK inhibi-tors such as trametinib (Mekinist GlaxoSmithKline) (NCT02224781) are now underway in melanoma Although a Phase I trial combining ipilimumab plus vemurafenib was terminated early owing to hepatotox-icity47 preliminary safety data from the combination trial of MEDI4736 (a PDL1-specific mAb developed by AstraZeneca) dabrafenib and trametinib appear promis-ing (n = 26) (NCT02027961)48

Combinations with VEGF-directed therapy The VEGFR tyrosine kinase inhibitor sunitinib blocks growth fac-tor signalling in tumour and vascular cells and also reduces the accumulation of MDSCs within tumours MDSCs are critical to the maintenance of the tumour microenvironment and are potent inhibitors of immune responses49 The impact of sunitinib on vascular cells and MDSCs is consistent with an impact on the tumour microenvironment a subject of substantial interest in rational immunotherapy design In studies of the effect of sunitinib on primary kidney cancer tumours 3 out of 8 kidney tumour specimens had low levels of MDSCs and normal T cell production of IFNγ whereas 5 out of 8 specimens showed high intratumoural MDSCs and low T cell production of IFNγ50 Such data have sup-ported the design of a Phase I trial combining nivolumab with either sunitinib or pazopanib as second-line ther-apy in patients with kidney cancer22 Notably response rates with the Phase I combinations were greater than expected for each agent individually22 Increased grade 3ndash4 toxicities observed with this combination were largely reversible by treatment with steroids or delaying or stopping further drug doses

Targeting VEGF directly may result in a more effec-tive combination than inhibiting the receptor Kidney cancers that progress on VEGFR tyrosine kinase inhibi-tor therapy are associated with increased VEGF produc-tion by the tumour51 Many tumour types also secrete VEGF and elevated VEGF serum levels are a marker of poor prognosis in diverse cancer indications52ndash54 VEGF can be immunosuppressive because its natural role is to support tissue remodelling and repair For example by removing signalling through VEGFR VEGF blockade can enhance dendritic cell function and subsequent T cell activation5556 Thus it is promising that the combination of the VEGF-specific mAb bevacizumab (Avastin GenentechRoche) and the PDL1-specific mAb atezolizumab (formerly known as MPDL3280A developed by GenentechRoche) has demonstrated only modest toxicity in Phase I trials57 Such observations pro-vide a rationale for why this combination therapy may be more effective in kidney cancer than monotherapy either as first-line therapy or after progression (when VEGF secretion is upregulated)

VEGF has many functions in addition to angio-genesis and can signal through receptors other than VEGF tyrosine kinase receptors For example neuro-pilins are multifunctional non-tyrosine kinase recep-tors that bind to specific isoforms of VEGF and to TGFβ and semaphorins58 Semaphorins are linked to tumour proliferation and survival in addition to their role in axonal guidance Neuropilin is also expressed on a subset of human TReg cells (FIG 3) and on a specialized subset of dendritic cells termed plasmacytoid dendritic cells although the functional relevance of this expres-sion in the immune system is unclear5960 Neuropilin is upregulated in multiple tumour types and expres-sion correlates with tumour progression61 A Phase I trial of MNRP1685A (a human immunoglobulin G1 (IgG1) mAb developed by Roche) which targets the VEGF-binding domain of neuropilin 1 demonstrated tolerability62 However a Phase Ib trial combining an

Other

Phosphatidylserine Bavituximab Phase IIIII

SIRPAndashCD47 CC-90002 Phase I

VEGF Bevacizumab FDA approved

Neuropilin MNRP1685A Phase I

BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CSF1R macrophage colony-stimulating factor receptor 1 CTLA4 cytotoxic T lymphocyte antigen 4 CXCL12 chemokine (C-X-C motif) ligand 12 CXCR4 C-X-C chemokine receptor type 4 GITR glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator IDO indoleamine 23-dioxygenase IgSF immunoglobulin superfamily ILT immunoglobulin-like transcript KIR killer inhibitory immunoglobulin-like receptor LAG3 lymphocyte activation gene 3 protein LIR leukocyte immunoglobulin-like LTA lymphotoxin-α MIC MHC class I polypeptide-related sequence PD1 programmed cell death protein 1 PDL1 programmed cell death 1 ligand 1 PVR poliovirus receptor SIRPA signal-regulatory protein- TGFβ transforming growth factor-β TIGIT T cell immunoreceptor with immunoglobulin and ITIM domains TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 TNFRSF25 TNFR superfamily member 25 TNFR TNF receptor VEGF vascular endothelial growth factor VISTA V-domain immunoglobulin suppressor of T cell activation Plerixafor (Mozobil GenzymeSanofi) is approved by the US Food and Drug Administration not as an antitumour therapy but as a bone marrow mobilizing agent for bone marrow transplantation

Table 1 (cont) | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

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TNF receptors(TNFRs) A large family of proteins associated with diverse cellular activities including the activation of lymphocytes

TNFR superfamily(TNFRSF) A group containing approximately 30 type I or type III membrane proteins and several secreted proteins TNFRSF receptors are characterized by the presence of one to four extracellular cysteine-rich ligand-binding domains

anti-neuropilin agent the VEGF-specific mAb beva-cizumab and chemotherapy resulted in greater than expected proteinuria63 This result suggests that dual blockade of both VEGF and neuropilin may be lim-ited by toxicity However the future development of a neuropilin-specific mAb may have a role independent of VEGF function by targeting the immune response through TReg cells The recent finding that semaphorin 4A signals through neuropilin 1 to regulate TReg cell function in the tumour environment suggests that tar-geting this pathway may deactivate TReg cells facilitat-ing the development of antitumour immunity64 (FIG 3) The role of neuropilin 1 on TReg cells is being intensively investigated with the hope that inhibiting this activity of neuropilin 1 can selectively target TReg cells

New targets the TNFR superfamilyConcurrent with the discovery and targeting of CD28 and CTLA4 members of the tumour necrosis factor (TNF) family of proteins were identified and developed as some of the first molecularly defined immunothera-peutics Local infusion of TNF and lymphotoxin-α has

been used as a treatment for primary melanoma and sarcoma for decades although with limited efficacy and pronounced toxicity65 Other early attempts targeted various TNF receptors (TNFRs) with agonist antibodies to directly induce tumour cell death Examples of this class of therapeutics include antibodies directed against TRAIL receptor 2 (also known as TNFRSF10B) lymphotoxin-β receptor (also known as TNFRSF3) and FN14 (also known as TNFRSF12A) Some of these approaches were limited by toxicity notably hepatic toxicity The next wave of antibody development target-ing this receptor family came with the identification of 4-1BB (also known as CD137TNFRSF9) and related TNFRs as critical regulators of previously activated T cells66 There are six receptors in the TNFR superfamily (TNFRSF) currently receiving attention as immune-activating agents these are discussed below In contrast to immune checkpoint targets such as PD1 the goal of most of these antibodies is to activate the receptors thus they act as agonists (FIG 1) The driving concept for com-bination therapy is that TNFRSF agonists will further activate and support the antitumour immune response released by immune checkpoint inhibition

4-1BB 4-1BB is a co-stimulatory receptor on T cells and natural killer cells and it is expressed on neutrophils and myeloid lineage cells including some subpopula-tions of dendritic cells Ligation of 4-1BB on T cells and natural killer cells induces cell activation survival and enhances effector functions 4-1BB is expressed rapidly after T cell activation and its expression continues for up to 48 hours 4-1BB is found on all activated T cell subsets including TReg cells The pleiotropic activity of 4-1BB agonist antibodies is apparent in preclinical studies in which the effects varied depending on the models Preclinical data demonstrated 4-1BB effects on CD8+ T cell proliferation and survival In some model systems effects on CD4+ T cells have been reported The role of 4-1BB in TReg cell function is confusing as conflicting data suggest that 4-1BB activity can either expand or restrict TReg cell activation Importantly 41bb knockout mice can develop autoimmunity suggesting a role for 4-1BB in maintaining immune homeostasis67 Furthermore 41bb knockout mice are deficient in their ability to reject tumours in tumour challenge mod-els demonstrating that effective antitumour memory requires 4-1BB Some 4-1BB-deficient mice spontane-ously develop B cell lymphomas as they age but whether this is a consequence of underlying proliferation of auto-immune B cells or defective immunoediting is unclear

Two agonistic 4-1BB-specific antibodies urelumab (an IgG4 mAb developed by Bristol-Meyers Squibb) and PF-05082566 (an IgG2 mAb developed by Pfizer) are in Phase I trials Preliminary findings support the hypoth-esis that 4-1BB-specific agonist activity will increase T cell and natural killer cell proliferation and activity68 A Phase II trial of urelumab in patients with melanoma was terminated early owing to a high incidence of grade 4 hepatotoxicity69 New trials with urelumab are under-way to establish a safe and efficacious dose includ-ing a monotherapy trial in patients with advanced or

Box 2 | Lessons learnt from CTLA4 inhibitors

In both preclinical and clinical models cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors were developed in combination with other anticancer therapies Anti-CTLA4 monotherapy was not effective in the poorly immunogenic B16 melanoma mouse model however the combination of a CTLA4 inhibitor with a granulocytendashmacrophage colony stimulating factor (GM-CSF)-producing tumour vaccine resulted in tumour rejection as well as immune-related vitiligo215 Surviving mice also rejected tumour re-challenge supporting the hypothesis that CTLA4 blockade can support the development of antitumour immune memory In clinical trials the combination of ipilimumab with the gp100 peptide vaccine did not improve the clinical benefit of ipilimumab6 Moreover in a Phase II trial of high-dose ipilimumab (10 mg per kg) and GM-CSF greater antitumour responses were observed and with less toxicity than with ipilimumab alone216 It is unclear whether GM-CSF is playing a protective role that limits toxicity Preclinical studies have shown that GM-CSF can recruit dendritic cells but also enhance regulatory T (T

Reg) cells

providing possible mechanisms for enhanced activity and reduced toxicity217 These results successfully informed the design of a Phase III trial

It is clear that development of anti-CTLA4 combination therapies have been limited by toxicity as seen in Phase I trials that combine CTLA4 inhibitors with small-molecule agents For example adding ipilimumab after 1 month of treatment with the BRAF inhibitor vemurafenib caused undue toxicity Eight of the first 10 patients enrolled developed asymptomatic grade 3 liver toxicity within 5 weeks of starting ipilimumab which was reversible with drug discontinuation or active suppression of the immune response with glucocorticoid steroids47 Similarly the dose-limiting toxicity observed in the combination trial of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor sunitinib with the CTLA4-blocking antibody tremelimumab was rapid-onset acute kidney injury that was reversible with steroids218 Both trials were terminated due to toxicity Separate from its toxicity profile mechanistically anti-CTLA4 therapy may not be an optimal backbone for improving efficacy with other therapies When ipilimumab was combined with high-dose IL-2 ipilimumab failed to show any synergy producing only a 22 response rate little more than either therapy alone219 Similarly the Phase III trial combining ipilimumab with the chemotherapy dacarbazine demonstrated that ipilimumab in combination with dacarbazine provided an improved survival benefit over dacarbazine alone but the survival rate was comparable with subsequent trials evaluating ipilimumab as monotherapy6220 Although some combinations may fail to synergize the antitumour effect others may even blunt the antitumour effect Therefore given the multitude of potential combinations rational design is essential

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Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

580 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 4: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

SuperagonistAn agent that produces a supra-physiological response greater than the response seen in normal physiology

Co-stimulatory signalA second non-antigen-specific signal that works with T cell receptor signalling to increase T cell activation

Checkpoint inhibition why combinationsA subset of patients with advanced cancers can respond to single-agent immune checkpoint blockade but most patients do not respond to such single-agent therapy Predictive biomarkers may provide a means to identify which patients will respond to monotherapy (BOX 4) Combining immunological agents may improve response rates and also improve the duration of response by stimulating an antitumour immunological memory Combinations of immunotherapies will require carefully planned Phase I dose-finding trials to assess the danger of overstimulating the immune system Such danger was illustrated by the clinical experience of targeting the T cell co-stimulatory receptor CD28 (REF 18) wherein 6 out of 6 subjects treated with a CD28 superagonist antibody developed life-threatening toxicity (a cytokine storm) in a Phase I trial leading to significant reluctance to further develop CD28 stimulatory agents19

Encouraging results from combinations with PD1 and PDL1 inhibitors have been reported from multi-ple Phase I trials including combinations with CTLA4 blockers with cytotoxic chemotherapy with radiation therapy or with small-molecule inhibitors such as the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors sunitinib (Sutent Pfizer) or pazopanib (Votrient Novartis)1620ndash26 Although toxici-ties appear to occur more frequently with such combina-tions the most significant toxicities are immune-related adverse effects which can be severe but largely manage-able with immunosuppressants Numerous clinical trials are underway to assess the safety and efficacy of novel

combinations However given the expense of Phase III trials careful development and rational design of combi-nation therapies during Phase III trials will be necessary to best incorporate the new therapeutics into current treatment algorithms and to determine the optimal timing and sequencing of regimens The underlying sci-entific rationale of each of the key therapeutics discussed herein can guide such rational design

Checkpoint blockade as a backboneThe rationale for combining CTLA4 and PD1 blockers is strong because although both CTLA4 and PD1 are expressed on T lymphocytes these pathways have dif-ferent mechanisms for inhibiting the function of these cells32728 CTLA4 competes with CD28 for CD80CD86 ligands and thereby blocks the CD28 co-stimulatory signal that is necessary for robust T cell activation and effector function By contrast PD1 is expressed on activated lym-phocytes and overexpressed on exhausted lymphocytes29 The interaction between PD1 and its ligands reduces T cell activation and decreases their cytotoxic activity

In a trial comparing the PD1-blocking mAb nivolumab to the alkylating agent dacarbazine in patients with previously untreated metastatic melanoma nivolumab elicited greater objective response rates (40 versus 139 P lt 0001) progression-free survival and overall survival30 A Phase I trial combining nivolumab and ipilimumab reported an objective response rate of 5316 and an even better response was observed in a randomized Phase II clinical trial comparing the com-bination versus ipilimumab alone (61 versus 11

Nature Reviews | Drug Discovery

CD30

CD70

HHLA2

ICOSL

MHC class I

PDL1

PDL2

B7-H3

B7-H4

Butyrophilin family

Galectin

Phosphatidylserine

Tumour cell Tumor Expression

Glioblastoma melanoma

Kidney prostate pancreatic glioblastoma

Breast renal cell ovarian oesophageal gastric pancreatic melanoma among others

Prostate renal cell non-small cell lung pancreatic gastric ovarian colorectal urothelial cell among others

Oesophageal ovarian pancreatic hepatocellular breast Hodgkin mediastinal large B-cell lymphoma among others

Melanoma renal cell head and neck cervical glioblastoma bladder oesophageal breast hepatocellular Hodgkin lymphoma mediastinallarge B-cell lymphoma among others

Non-Hodgkin lymphoma renal cell

Hodgkin lymphoma embryonal anaplastic large cell lymphoma

Non-small cell lung colorectal gastric among others

Breast lung thyroid melanoma pancreas ovary liver bladder colon prostate kidney oesophagus

Refs

189 196 223230-232

CD155

Ligands

PDL1

PDL2

B7-H3

B7-H4

HHLA2

Galectins

CD30

CD70

ICOSL

CD155

233ndash238

239ndash246

247ndash257

129

258ndash261

262

263 264

265 266

267

Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligands Individual tumours can express a selected subset of co-inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL inducible T cell co-stimulator ligand PDL programmed cell death 1 ligand

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Myeloid-derived suppressor cells(MDSCs) A heterogeneous population of myeloid lineage cells with immunosuppresive activities

Histone deactylaseAn intracellular protein that regulates gene transcription by modifying histones and thereby chromosome structure

BRAFA serinethreonine kinase often associated with solid tumour oncogenesis and resistance to therapy mutant BRAF may be an effective target for therapeutics as seen with vemurafenib in V600E-mutated melanoma

Cross-presentationThe ability of some antigen presenting cells to take up extracellular proteins or cells and present their antigens in the context of MHC class I

P lt 0001) in patients with previously untreated meta-static melanoma17 A Phase III trial comparing mono-therapy nivolumab and monotherapy ipilimumab to the combination in patients with melanoma found that nivolumab alone was less toxic and had greater clinical benefit than ipilimumab alone (ClinicalTrialsgov identi-fier NCT01844505 note that all trials cited in this arti-cle are from this database (see Further information)14 While the overall survival results are pending in this study nivolumab as monotherapy and in combination with ipilimumab had better objective response rates than ipilimumab alone (437 576 and 19 respectively) Moreover there is an ongoing clinical trial comparing the sequence of nivolumab and ipilimumab (NCT01783938) to further investigate the toxicities of these treatments The favourable efficacy and tolerability profile of the PD1 pathway-blocking agents has generated enthusi-asm for their combination with other FDA-approved antitumour agents including small-molecule targeting agents standard chemotherapy and radiation therapy31 Compared to CTLA4 the PD1 pathway has a more subtle role in maintaining peripheral tolerance and regulating inflammation32 Thus PD1ndashPDL1 blockade has taken the lead as the foundation of combination therapy in current clinical trials

Combinations with chemotherapy Historically immu-notherapy was considered to be most effective in cases in which there is a small burden of tumour this reason-ing also appears to be true for newer agents7 However accumulating evidence indicates that immunotherapies are effective in a broad range of tumours and therefore combining chemotherapy with immune checkpoint inhibitors may take advantage of three effects of chemo-therapy First to reduce the tumour burden second to potentiate the antitumour response by exposing neoanti-gen via necrosis of the tumour or third to directly affect the tumour stromal cells The choice of chemothera-peutic agent and timing of these combinations will be important because many cytotoxic chemotherapeutics target rapidly dividing cells Chemotherapy regimens that deplete proliferating lymphocytes may negatively affect the efficacy of therapeutics such as ipilimumab and nivolumab which act by facilitating the activa-tion and proliferation of tumour-infiltrating lympho-cytes Indeed increased absolute lymphocyte count may be a pharmacodynamic marker of the response to ipilimumab33

There is intriguing data to support the hypothesis that cytotoxic chemotherapy alters the immunosup-pressive microenvironment of the tumour Historically cyclophosphamide has been used to deplete TReg cells in preclinical adoptive T cell and vaccine models34 and this regimen may augment immunotherapies in patients Alternatively various chemotherapies such as 5-fluoro-uracil gemcitabine and taxanes can cause a decrease in myeloid-derived suppressor cells (MDSCs)3536 Some chemotherapy regimens may prime the immune sys-tem to better respond to immune checkpoint blockade Substantial data support the hypothesis that some chem-otherapeutic agents may function as a vaccine killing

tumour cells and increasing the amount of tumour antigen processed and presented to T cells (reviewed in REF 37) Chemotherapeutic agents that target epigenetic modifiers (for example histone deacetylase) are able to synergize with CTLA4 or PD1 blockers to eradicate pri-mary tumour and metastases in murine models of large established tumours that are poorly immunogenic38

Caution is warranted in developing combina-tion trials because the efficacy of standard therapy may be negatively affected Fortunately early reports of nivolumab in combination with standard-of-care platinum-based chemotherapy couplets (for exam-ple gemcitabinendashcisplatin pemetrexedndashcisplatin and paclitaxelndashcarboplatin) in a Phase I NSCLC trial have shown overall response rates similar to historical con-trols21 Discontinuation of therapy owing to treatment-related adverse effects was observed in 20 of patients Longer follow-up is necessary to determine whether the responses from these initial combination trials in NSCLC are more durable than standard chemotherapy Notably platinum-based chemotherapy causes sig-nificant nausea and taxanes can produce significant adverse reactions thus pretreatment with steroids is standard It is unclear how concurrent corticosteroid use will affect the efficacy of immune checkpoint inhibi-tors In mice corticosteroid administration abrogates the antitumour effect of lymphokine-activated killer cells and IL-2 (REF 39) In patients with advanced can-cer dexamethasone significantly reduced the toxicity of high-dose IL-2 but also significantly reduced the response rate to this potentially curative regimen4041 Although steroids are highly successful at reversing immune-related adverse effects due to immune check-point inhibitors the results with IL-2 and steroids raise concern that concurrent use will negatively affect the efficacy of other immunotherapies Although immune-related adverse effects are observed in patients treated with immune checkpoint blockers when steroids are used to treat such adverse effects they do not appear to abrogate the clinical benefit of treatment1417 Indeed a substantial number of patients in a study of ipilimumab plus nivolumab required immunosuppressive agents to treat toxicity (834) however of those who withdrew from the study owing to toxicity many responded to the combination (675 81 out of 120 patients)1442 A series of randomized clinical trials in NCSLC will be needed to determine the role of immune checkpoint blockade in combination or following the current standard therapy

Combinations with BRAF inhibitors With the renewed interest in immunotherapy the mechanism of action and resistance of many molecularly targeted agents is being re-examined and has been found to include immunological effects Treatment with the BRAF inhibi-tor vemurafenib (Zelboraf Roche) appears to improve the antitumour immune response to melanoma perhaps by increasing the cross-presentation of antigens from dead tumour cells4344 The development of resistance to BRAF inhibitors is accompanied by an increased expres-sion of PDL1 on the melanoma cells45 Studies using a mouse model of BRAFV600E mutant melanoma showed

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Table 1 | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

CTLA4 Ipilimumab FDA approved

Tremelimumab Phase III

PD1ndashPDL1 Pembrolizumab (PD1) FDA approved

Nivolumab (PD1) FDA approved

Atezolizumab (formerly MPDL3280A) (PDL1) Phase III

MEDI4736 (PDL1) Phase III

Avelumab (PDL1) Phase I

PDR001 (PD1) Phase I

TNF and TNFR superfamilies

4-1BBndash4-1BB ligand Urelumab PF-05082566 Phase II

OX40ndashOX40 ligand MEDI6469 Phase II

GITR TRX518 Phase I

CD27 Varlilumab Phase II

TNFRSF25ndashTL1A Preclinical

CD40ndashCD40 ligand CP-870893 Phase I

HVEMndashLIGHTndashLTA Preclinical

HVEMndashBTLAndashCD160 Preclinical

IgSF

LAG3 BMS-986016 Phase I

TIM3 Preclinical

Siglecs Preclinical

B7 and CD28‑related proteins

ICOSndashICOS ligand Preclinical

B7-H3 MGA271 Phase I

B7-H4 Preclinical

VISTA Preclinical

HHLA2ndashTMIGD2 Preclinical

Butyrophilins including BTNL2 Preclinical

CD244ndashCD48 Preclinical

TIGIT and PVR family members Preclinical

Natural killer cell targets

KIRs Lirilumab Phase II

ILTs and LIRs Preclinical

NKG2D and NKG2A IPH2201 Phase I

MICA and MICB Preclinical

CD244 Preclinical

Suppressive myeloid cells

CSF1R Emactuzumab Phase I

Soluble mediators

IDO INCB024360 Phase II

TGFβ Galunisertib Phase I

AdenosinendashCD39ndashCD73 Preclinical

CXCR4ndashCXCL12 Ulocuplumab BKT140 Phase III

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that PD1 or PDL1 blockade combined with BRAF inhibition increased the activity of tumour -infiltrating lymphocytes and prolonged survival46 Based on such observations clinical trials evaluating combinations of PD1 pathway inhibitors with BRAF inhibitors such as vemurafenib (NCT01656642) or dabrafenib (Tafinlar GlaxoSmithKline) and with MEK inhibi-tors such as trametinib (Mekinist GlaxoSmithKline) (NCT02224781) are now underway in melanoma Although a Phase I trial combining ipilimumab plus vemurafenib was terminated early owing to hepatotox-icity47 preliminary safety data from the combination trial of MEDI4736 (a PDL1-specific mAb developed by AstraZeneca) dabrafenib and trametinib appear promis-ing (n = 26) (NCT02027961)48

Combinations with VEGF-directed therapy The VEGFR tyrosine kinase inhibitor sunitinib blocks growth fac-tor signalling in tumour and vascular cells and also reduces the accumulation of MDSCs within tumours MDSCs are critical to the maintenance of the tumour microenvironment and are potent inhibitors of immune responses49 The impact of sunitinib on vascular cells and MDSCs is consistent with an impact on the tumour microenvironment a subject of substantial interest in rational immunotherapy design In studies of the effect of sunitinib on primary kidney cancer tumours 3 out of 8 kidney tumour specimens had low levels of MDSCs and normal T cell production of IFNγ whereas 5 out of 8 specimens showed high intratumoural MDSCs and low T cell production of IFNγ50 Such data have sup-ported the design of a Phase I trial combining nivolumab with either sunitinib or pazopanib as second-line ther-apy in patients with kidney cancer22 Notably response rates with the Phase I combinations were greater than expected for each agent individually22 Increased grade 3ndash4 toxicities observed with this combination were largely reversible by treatment with steroids or delaying or stopping further drug doses

Targeting VEGF directly may result in a more effec-tive combination than inhibiting the receptor Kidney cancers that progress on VEGFR tyrosine kinase inhibi-tor therapy are associated with increased VEGF produc-tion by the tumour51 Many tumour types also secrete VEGF and elevated VEGF serum levels are a marker of poor prognosis in diverse cancer indications52ndash54 VEGF can be immunosuppressive because its natural role is to support tissue remodelling and repair For example by removing signalling through VEGFR VEGF blockade can enhance dendritic cell function and subsequent T cell activation5556 Thus it is promising that the combination of the VEGF-specific mAb bevacizumab (Avastin GenentechRoche) and the PDL1-specific mAb atezolizumab (formerly known as MPDL3280A developed by GenentechRoche) has demonstrated only modest toxicity in Phase I trials57 Such observations pro-vide a rationale for why this combination therapy may be more effective in kidney cancer than monotherapy either as first-line therapy or after progression (when VEGF secretion is upregulated)

VEGF has many functions in addition to angio-genesis and can signal through receptors other than VEGF tyrosine kinase receptors For example neuro-pilins are multifunctional non-tyrosine kinase recep-tors that bind to specific isoforms of VEGF and to TGFβ and semaphorins58 Semaphorins are linked to tumour proliferation and survival in addition to their role in axonal guidance Neuropilin is also expressed on a subset of human TReg cells (FIG 3) and on a specialized subset of dendritic cells termed plasmacytoid dendritic cells although the functional relevance of this expres-sion in the immune system is unclear5960 Neuropilin is upregulated in multiple tumour types and expres-sion correlates with tumour progression61 A Phase I trial of MNRP1685A (a human immunoglobulin G1 (IgG1) mAb developed by Roche) which targets the VEGF-binding domain of neuropilin 1 demonstrated tolerability62 However a Phase Ib trial combining an

Other

Phosphatidylserine Bavituximab Phase IIIII

SIRPAndashCD47 CC-90002 Phase I

VEGF Bevacizumab FDA approved

Neuropilin MNRP1685A Phase I

BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CSF1R macrophage colony-stimulating factor receptor 1 CTLA4 cytotoxic T lymphocyte antigen 4 CXCL12 chemokine (C-X-C motif) ligand 12 CXCR4 C-X-C chemokine receptor type 4 GITR glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator IDO indoleamine 23-dioxygenase IgSF immunoglobulin superfamily ILT immunoglobulin-like transcript KIR killer inhibitory immunoglobulin-like receptor LAG3 lymphocyte activation gene 3 protein LIR leukocyte immunoglobulin-like LTA lymphotoxin-α MIC MHC class I polypeptide-related sequence PD1 programmed cell death protein 1 PDL1 programmed cell death 1 ligand 1 PVR poliovirus receptor SIRPA signal-regulatory protein- TGFβ transforming growth factor-β TIGIT T cell immunoreceptor with immunoglobulin and ITIM domains TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 TNFRSF25 TNFR superfamily member 25 TNFR TNF receptor VEGF vascular endothelial growth factor VISTA V-domain immunoglobulin suppressor of T cell activation Plerixafor (Mozobil GenzymeSanofi) is approved by the US Food and Drug Administration not as an antitumour therapy but as a bone marrow mobilizing agent for bone marrow transplantation

Table 1 (cont) | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

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TNF receptors(TNFRs) A large family of proteins associated with diverse cellular activities including the activation of lymphocytes

TNFR superfamily(TNFRSF) A group containing approximately 30 type I or type III membrane proteins and several secreted proteins TNFRSF receptors are characterized by the presence of one to four extracellular cysteine-rich ligand-binding domains

anti-neuropilin agent the VEGF-specific mAb beva-cizumab and chemotherapy resulted in greater than expected proteinuria63 This result suggests that dual blockade of both VEGF and neuropilin may be lim-ited by toxicity However the future development of a neuropilin-specific mAb may have a role independent of VEGF function by targeting the immune response through TReg cells The recent finding that semaphorin 4A signals through neuropilin 1 to regulate TReg cell function in the tumour environment suggests that tar-geting this pathway may deactivate TReg cells facilitat-ing the development of antitumour immunity64 (FIG 3) The role of neuropilin 1 on TReg cells is being intensively investigated with the hope that inhibiting this activity of neuropilin 1 can selectively target TReg cells

New targets the TNFR superfamilyConcurrent with the discovery and targeting of CD28 and CTLA4 members of the tumour necrosis factor (TNF) family of proteins were identified and developed as some of the first molecularly defined immunothera-peutics Local infusion of TNF and lymphotoxin-α has

been used as a treatment for primary melanoma and sarcoma for decades although with limited efficacy and pronounced toxicity65 Other early attempts targeted various TNF receptors (TNFRs) with agonist antibodies to directly induce tumour cell death Examples of this class of therapeutics include antibodies directed against TRAIL receptor 2 (also known as TNFRSF10B) lymphotoxin-β receptor (also known as TNFRSF3) and FN14 (also known as TNFRSF12A) Some of these approaches were limited by toxicity notably hepatic toxicity The next wave of antibody development target-ing this receptor family came with the identification of 4-1BB (also known as CD137TNFRSF9) and related TNFRs as critical regulators of previously activated T cells66 There are six receptors in the TNFR superfamily (TNFRSF) currently receiving attention as immune-activating agents these are discussed below In contrast to immune checkpoint targets such as PD1 the goal of most of these antibodies is to activate the receptors thus they act as agonists (FIG 1) The driving concept for com-bination therapy is that TNFRSF agonists will further activate and support the antitumour immune response released by immune checkpoint inhibition

4-1BB 4-1BB is a co-stimulatory receptor on T cells and natural killer cells and it is expressed on neutrophils and myeloid lineage cells including some subpopula-tions of dendritic cells Ligation of 4-1BB on T cells and natural killer cells induces cell activation survival and enhances effector functions 4-1BB is expressed rapidly after T cell activation and its expression continues for up to 48 hours 4-1BB is found on all activated T cell subsets including TReg cells The pleiotropic activity of 4-1BB agonist antibodies is apparent in preclinical studies in which the effects varied depending on the models Preclinical data demonstrated 4-1BB effects on CD8+ T cell proliferation and survival In some model systems effects on CD4+ T cells have been reported The role of 4-1BB in TReg cell function is confusing as conflicting data suggest that 4-1BB activity can either expand or restrict TReg cell activation Importantly 41bb knockout mice can develop autoimmunity suggesting a role for 4-1BB in maintaining immune homeostasis67 Furthermore 41bb knockout mice are deficient in their ability to reject tumours in tumour challenge mod-els demonstrating that effective antitumour memory requires 4-1BB Some 4-1BB-deficient mice spontane-ously develop B cell lymphomas as they age but whether this is a consequence of underlying proliferation of auto-immune B cells or defective immunoediting is unclear

Two agonistic 4-1BB-specific antibodies urelumab (an IgG4 mAb developed by Bristol-Meyers Squibb) and PF-05082566 (an IgG2 mAb developed by Pfizer) are in Phase I trials Preliminary findings support the hypoth-esis that 4-1BB-specific agonist activity will increase T cell and natural killer cell proliferation and activity68 A Phase II trial of urelumab in patients with melanoma was terminated early owing to a high incidence of grade 4 hepatotoxicity69 New trials with urelumab are under-way to establish a safe and efficacious dose includ-ing a monotherapy trial in patients with advanced or

Box 2 | Lessons learnt from CTLA4 inhibitors

In both preclinical and clinical models cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors were developed in combination with other anticancer therapies Anti-CTLA4 monotherapy was not effective in the poorly immunogenic B16 melanoma mouse model however the combination of a CTLA4 inhibitor with a granulocytendashmacrophage colony stimulating factor (GM-CSF)-producing tumour vaccine resulted in tumour rejection as well as immune-related vitiligo215 Surviving mice also rejected tumour re-challenge supporting the hypothesis that CTLA4 blockade can support the development of antitumour immune memory In clinical trials the combination of ipilimumab with the gp100 peptide vaccine did not improve the clinical benefit of ipilimumab6 Moreover in a Phase II trial of high-dose ipilimumab (10 mg per kg) and GM-CSF greater antitumour responses were observed and with less toxicity than with ipilimumab alone216 It is unclear whether GM-CSF is playing a protective role that limits toxicity Preclinical studies have shown that GM-CSF can recruit dendritic cells but also enhance regulatory T (T

Reg) cells

providing possible mechanisms for enhanced activity and reduced toxicity217 These results successfully informed the design of a Phase III trial

It is clear that development of anti-CTLA4 combination therapies have been limited by toxicity as seen in Phase I trials that combine CTLA4 inhibitors with small-molecule agents For example adding ipilimumab after 1 month of treatment with the BRAF inhibitor vemurafenib caused undue toxicity Eight of the first 10 patients enrolled developed asymptomatic grade 3 liver toxicity within 5 weeks of starting ipilimumab which was reversible with drug discontinuation or active suppression of the immune response with glucocorticoid steroids47 Similarly the dose-limiting toxicity observed in the combination trial of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor sunitinib with the CTLA4-blocking antibody tremelimumab was rapid-onset acute kidney injury that was reversible with steroids218 Both trials were terminated due to toxicity Separate from its toxicity profile mechanistically anti-CTLA4 therapy may not be an optimal backbone for improving efficacy with other therapies When ipilimumab was combined with high-dose IL-2 ipilimumab failed to show any synergy producing only a 22 response rate little more than either therapy alone219 Similarly the Phase III trial combining ipilimumab with the chemotherapy dacarbazine demonstrated that ipilimumab in combination with dacarbazine provided an improved survival benefit over dacarbazine alone but the survival rate was comparable with subsequent trials evaluating ipilimumab as monotherapy6220 Although some combinations may fail to synergize the antitumour effect others may even blunt the antitumour effect Therefore given the multitude of potential combinations rational design is essential

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Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 577

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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copy 2015 Macmillan Publishers Limited All rights reserved

antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 5: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

Myeloid-derived suppressor cells(MDSCs) A heterogeneous population of myeloid lineage cells with immunosuppresive activities

Histone deactylaseAn intracellular protein that regulates gene transcription by modifying histones and thereby chromosome structure

BRAFA serinethreonine kinase often associated with solid tumour oncogenesis and resistance to therapy mutant BRAF may be an effective target for therapeutics as seen with vemurafenib in V600E-mutated melanoma

Cross-presentationThe ability of some antigen presenting cells to take up extracellular proteins or cells and present their antigens in the context of MHC class I

P lt 0001) in patients with previously untreated meta-static melanoma17 A Phase III trial comparing mono-therapy nivolumab and monotherapy ipilimumab to the combination in patients with melanoma found that nivolumab alone was less toxic and had greater clinical benefit than ipilimumab alone (ClinicalTrialsgov identi-fier NCT01844505 note that all trials cited in this arti-cle are from this database (see Further information)14 While the overall survival results are pending in this study nivolumab as monotherapy and in combination with ipilimumab had better objective response rates than ipilimumab alone (437 576 and 19 respectively) Moreover there is an ongoing clinical trial comparing the sequence of nivolumab and ipilimumab (NCT01783938) to further investigate the toxicities of these treatments The favourable efficacy and tolerability profile of the PD1 pathway-blocking agents has generated enthusi-asm for their combination with other FDA-approved antitumour agents including small-molecule targeting agents standard chemotherapy and radiation therapy31 Compared to CTLA4 the PD1 pathway has a more subtle role in maintaining peripheral tolerance and regulating inflammation32 Thus PD1ndashPDL1 blockade has taken the lead as the foundation of combination therapy in current clinical trials

Combinations with chemotherapy Historically immu-notherapy was considered to be most effective in cases in which there is a small burden of tumour this reason-ing also appears to be true for newer agents7 However accumulating evidence indicates that immunotherapies are effective in a broad range of tumours and therefore combining chemotherapy with immune checkpoint inhibitors may take advantage of three effects of chemo-therapy First to reduce the tumour burden second to potentiate the antitumour response by exposing neoanti-gen via necrosis of the tumour or third to directly affect the tumour stromal cells The choice of chemothera-peutic agent and timing of these combinations will be important because many cytotoxic chemotherapeutics target rapidly dividing cells Chemotherapy regimens that deplete proliferating lymphocytes may negatively affect the efficacy of therapeutics such as ipilimumab and nivolumab which act by facilitating the activa-tion and proliferation of tumour-infiltrating lympho-cytes Indeed increased absolute lymphocyte count may be a pharmacodynamic marker of the response to ipilimumab33

There is intriguing data to support the hypothesis that cytotoxic chemotherapy alters the immunosup-pressive microenvironment of the tumour Historically cyclophosphamide has been used to deplete TReg cells in preclinical adoptive T cell and vaccine models34 and this regimen may augment immunotherapies in patients Alternatively various chemotherapies such as 5-fluoro-uracil gemcitabine and taxanes can cause a decrease in myeloid-derived suppressor cells (MDSCs)3536 Some chemotherapy regimens may prime the immune sys-tem to better respond to immune checkpoint blockade Substantial data support the hypothesis that some chem-otherapeutic agents may function as a vaccine killing

tumour cells and increasing the amount of tumour antigen processed and presented to T cells (reviewed in REF 37) Chemotherapeutic agents that target epigenetic modifiers (for example histone deacetylase) are able to synergize with CTLA4 or PD1 blockers to eradicate pri-mary tumour and metastases in murine models of large established tumours that are poorly immunogenic38

Caution is warranted in developing combina-tion trials because the efficacy of standard therapy may be negatively affected Fortunately early reports of nivolumab in combination with standard-of-care platinum-based chemotherapy couplets (for exam-ple gemcitabinendashcisplatin pemetrexedndashcisplatin and paclitaxelndashcarboplatin) in a Phase I NSCLC trial have shown overall response rates similar to historical con-trols21 Discontinuation of therapy owing to treatment-related adverse effects was observed in 20 of patients Longer follow-up is necessary to determine whether the responses from these initial combination trials in NSCLC are more durable than standard chemotherapy Notably platinum-based chemotherapy causes sig-nificant nausea and taxanes can produce significant adverse reactions thus pretreatment with steroids is standard It is unclear how concurrent corticosteroid use will affect the efficacy of immune checkpoint inhibi-tors In mice corticosteroid administration abrogates the antitumour effect of lymphokine-activated killer cells and IL-2 (REF 39) In patients with advanced can-cer dexamethasone significantly reduced the toxicity of high-dose IL-2 but also significantly reduced the response rate to this potentially curative regimen4041 Although steroids are highly successful at reversing immune-related adverse effects due to immune check-point inhibitors the results with IL-2 and steroids raise concern that concurrent use will negatively affect the efficacy of other immunotherapies Although immune-related adverse effects are observed in patients treated with immune checkpoint blockers when steroids are used to treat such adverse effects they do not appear to abrogate the clinical benefit of treatment1417 Indeed a substantial number of patients in a study of ipilimumab plus nivolumab required immunosuppressive agents to treat toxicity (834) however of those who withdrew from the study owing to toxicity many responded to the combination (675 81 out of 120 patients)1442 A series of randomized clinical trials in NCSLC will be needed to determine the role of immune checkpoint blockade in combination or following the current standard therapy

Combinations with BRAF inhibitors With the renewed interest in immunotherapy the mechanism of action and resistance of many molecularly targeted agents is being re-examined and has been found to include immunological effects Treatment with the BRAF inhibi-tor vemurafenib (Zelboraf Roche) appears to improve the antitumour immune response to melanoma perhaps by increasing the cross-presentation of antigens from dead tumour cells4344 The development of resistance to BRAF inhibitors is accompanied by an increased expres-sion of PDL1 on the melanoma cells45 Studies using a mouse model of BRAFV600E mutant melanoma showed

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Table 1 | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

CTLA4 Ipilimumab FDA approved

Tremelimumab Phase III

PD1ndashPDL1 Pembrolizumab (PD1) FDA approved

Nivolumab (PD1) FDA approved

Atezolizumab (formerly MPDL3280A) (PDL1) Phase III

MEDI4736 (PDL1) Phase III

Avelumab (PDL1) Phase I

PDR001 (PD1) Phase I

TNF and TNFR superfamilies

4-1BBndash4-1BB ligand Urelumab PF-05082566 Phase II

OX40ndashOX40 ligand MEDI6469 Phase II

GITR TRX518 Phase I

CD27 Varlilumab Phase II

TNFRSF25ndashTL1A Preclinical

CD40ndashCD40 ligand CP-870893 Phase I

HVEMndashLIGHTndashLTA Preclinical

HVEMndashBTLAndashCD160 Preclinical

IgSF

LAG3 BMS-986016 Phase I

TIM3 Preclinical

Siglecs Preclinical

B7 and CD28‑related proteins

ICOSndashICOS ligand Preclinical

B7-H3 MGA271 Phase I

B7-H4 Preclinical

VISTA Preclinical

HHLA2ndashTMIGD2 Preclinical

Butyrophilins including BTNL2 Preclinical

CD244ndashCD48 Preclinical

TIGIT and PVR family members Preclinical

Natural killer cell targets

KIRs Lirilumab Phase II

ILTs and LIRs Preclinical

NKG2D and NKG2A IPH2201 Phase I

MICA and MICB Preclinical

CD244 Preclinical

Suppressive myeloid cells

CSF1R Emactuzumab Phase I

Soluble mediators

IDO INCB024360 Phase II

TGFβ Galunisertib Phase I

AdenosinendashCD39ndashCD73 Preclinical

CXCR4ndashCXCL12 Ulocuplumab BKT140 Phase III

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that PD1 or PDL1 blockade combined with BRAF inhibition increased the activity of tumour -infiltrating lymphocytes and prolonged survival46 Based on such observations clinical trials evaluating combinations of PD1 pathway inhibitors with BRAF inhibitors such as vemurafenib (NCT01656642) or dabrafenib (Tafinlar GlaxoSmithKline) and with MEK inhibi-tors such as trametinib (Mekinist GlaxoSmithKline) (NCT02224781) are now underway in melanoma Although a Phase I trial combining ipilimumab plus vemurafenib was terminated early owing to hepatotox-icity47 preliminary safety data from the combination trial of MEDI4736 (a PDL1-specific mAb developed by AstraZeneca) dabrafenib and trametinib appear promis-ing (n = 26) (NCT02027961)48

Combinations with VEGF-directed therapy The VEGFR tyrosine kinase inhibitor sunitinib blocks growth fac-tor signalling in tumour and vascular cells and also reduces the accumulation of MDSCs within tumours MDSCs are critical to the maintenance of the tumour microenvironment and are potent inhibitors of immune responses49 The impact of sunitinib on vascular cells and MDSCs is consistent with an impact on the tumour microenvironment a subject of substantial interest in rational immunotherapy design In studies of the effect of sunitinib on primary kidney cancer tumours 3 out of 8 kidney tumour specimens had low levels of MDSCs and normal T cell production of IFNγ whereas 5 out of 8 specimens showed high intratumoural MDSCs and low T cell production of IFNγ50 Such data have sup-ported the design of a Phase I trial combining nivolumab with either sunitinib or pazopanib as second-line ther-apy in patients with kidney cancer22 Notably response rates with the Phase I combinations were greater than expected for each agent individually22 Increased grade 3ndash4 toxicities observed with this combination were largely reversible by treatment with steroids or delaying or stopping further drug doses

Targeting VEGF directly may result in a more effec-tive combination than inhibiting the receptor Kidney cancers that progress on VEGFR tyrosine kinase inhibi-tor therapy are associated with increased VEGF produc-tion by the tumour51 Many tumour types also secrete VEGF and elevated VEGF serum levels are a marker of poor prognosis in diverse cancer indications52ndash54 VEGF can be immunosuppressive because its natural role is to support tissue remodelling and repair For example by removing signalling through VEGFR VEGF blockade can enhance dendritic cell function and subsequent T cell activation5556 Thus it is promising that the combination of the VEGF-specific mAb bevacizumab (Avastin GenentechRoche) and the PDL1-specific mAb atezolizumab (formerly known as MPDL3280A developed by GenentechRoche) has demonstrated only modest toxicity in Phase I trials57 Such observations pro-vide a rationale for why this combination therapy may be more effective in kidney cancer than monotherapy either as first-line therapy or after progression (when VEGF secretion is upregulated)

VEGF has many functions in addition to angio-genesis and can signal through receptors other than VEGF tyrosine kinase receptors For example neuro-pilins are multifunctional non-tyrosine kinase recep-tors that bind to specific isoforms of VEGF and to TGFβ and semaphorins58 Semaphorins are linked to tumour proliferation and survival in addition to their role in axonal guidance Neuropilin is also expressed on a subset of human TReg cells (FIG 3) and on a specialized subset of dendritic cells termed plasmacytoid dendritic cells although the functional relevance of this expres-sion in the immune system is unclear5960 Neuropilin is upregulated in multiple tumour types and expres-sion correlates with tumour progression61 A Phase I trial of MNRP1685A (a human immunoglobulin G1 (IgG1) mAb developed by Roche) which targets the VEGF-binding domain of neuropilin 1 demonstrated tolerability62 However a Phase Ib trial combining an

Other

Phosphatidylserine Bavituximab Phase IIIII

SIRPAndashCD47 CC-90002 Phase I

VEGF Bevacizumab FDA approved

Neuropilin MNRP1685A Phase I

BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CSF1R macrophage colony-stimulating factor receptor 1 CTLA4 cytotoxic T lymphocyte antigen 4 CXCL12 chemokine (C-X-C motif) ligand 12 CXCR4 C-X-C chemokine receptor type 4 GITR glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator IDO indoleamine 23-dioxygenase IgSF immunoglobulin superfamily ILT immunoglobulin-like transcript KIR killer inhibitory immunoglobulin-like receptor LAG3 lymphocyte activation gene 3 protein LIR leukocyte immunoglobulin-like LTA lymphotoxin-α MIC MHC class I polypeptide-related sequence PD1 programmed cell death protein 1 PDL1 programmed cell death 1 ligand 1 PVR poliovirus receptor SIRPA signal-regulatory protein- TGFβ transforming growth factor-β TIGIT T cell immunoreceptor with immunoglobulin and ITIM domains TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 TNFRSF25 TNFR superfamily member 25 TNFR TNF receptor VEGF vascular endothelial growth factor VISTA V-domain immunoglobulin suppressor of T cell activation Plerixafor (Mozobil GenzymeSanofi) is approved by the US Food and Drug Administration not as an antitumour therapy but as a bone marrow mobilizing agent for bone marrow transplantation

Table 1 (cont) | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

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TNF receptors(TNFRs) A large family of proteins associated with diverse cellular activities including the activation of lymphocytes

TNFR superfamily(TNFRSF) A group containing approximately 30 type I or type III membrane proteins and several secreted proteins TNFRSF receptors are characterized by the presence of one to four extracellular cysteine-rich ligand-binding domains

anti-neuropilin agent the VEGF-specific mAb beva-cizumab and chemotherapy resulted in greater than expected proteinuria63 This result suggests that dual blockade of both VEGF and neuropilin may be lim-ited by toxicity However the future development of a neuropilin-specific mAb may have a role independent of VEGF function by targeting the immune response through TReg cells The recent finding that semaphorin 4A signals through neuropilin 1 to regulate TReg cell function in the tumour environment suggests that tar-geting this pathway may deactivate TReg cells facilitat-ing the development of antitumour immunity64 (FIG 3) The role of neuropilin 1 on TReg cells is being intensively investigated with the hope that inhibiting this activity of neuropilin 1 can selectively target TReg cells

New targets the TNFR superfamilyConcurrent with the discovery and targeting of CD28 and CTLA4 members of the tumour necrosis factor (TNF) family of proteins were identified and developed as some of the first molecularly defined immunothera-peutics Local infusion of TNF and lymphotoxin-α has

been used as a treatment for primary melanoma and sarcoma for decades although with limited efficacy and pronounced toxicity65 Other early attempts targeted various TNF receptors (TNFRs) with agonist antibodies to directly induce tumour cell death Examples of this class of therapeutics include antibodies directed against TRAIL receptor 2 (also known as TNFRSF10B) lymphotoxin-β receptor (also known as TNFRSF3) and FN14 (also known as TNFRSF12A) Some of these approaches were limited by toxicity notably hepatic toxicity The next wave of antibody development target-ing this receptor family came with the identification of 4-1BB (also known as CD137TNFRSF9) and related TNFRs as critical regulators of previously activated T cells66 There are six receptors in the TNFR superfamily (TNFRSF) currently receiving attention as immune-activating agents these are discussed below In contrast to immune checkpoint targets such as PD1 the goal of most of these antibodies is to activate the receptors thus they act as agonists (FIG 1) The driving concept for com-bination therapy is that TNFRSF agonists will further activate and support the antitumour immune response released by immune checkpoint inhibition

4-1BB 4-1BB is a co-stimulatory receptor on T cells and natural killer cells and it is expressed on neutrophils and myeloid lineage cells including some subpopula-tions of dendritic cells Ligation of 4-1BB on T cells and natural killer cells induces cell activation survival and enhances effector functions 4-1BB is expressed rapidly after T cell activation and its expression continues for up to 48 hours 4-1BB is found on all activated T cell subsets including TReg cells The pleiotropic activity of 4-1BB agonist antibodies is apparent in preclinical studies in which the effects varied depending on the models Preclinical data demonstrated 4-1BB effects on CD8+ T cell proliferation and survival In some model systems effects on CD4+ T cells have been reported The role of 4-1BB in TReg cell function is confusing as conflicting data suggest that 4-1BB activity can either expand or restrict TReg cell activation Importantly 41bb knockout mice can develop autoimmunity suggesting a role for 4-1BB in maintaining immune homeostasis67 Furthermore 41bb knockout mice are deficient in their ability to reject tumours in tumour challenge mod-els demonstrating that effective antitumour memory requires 4-1BB Some 4-1BB-deficient mice spontane-ously develop B cell lymphomas as they age but whether this is a consequence of underlying proliferation of auto-immune B cells or defective immunoediting is unclear

Two agonistic 4-1BB-specific antibodies urelumab (an IgG4 mAb developed by Bristol-Meyers Squibb) and PF-05082566 (an IgG2 mAb developed by Pfizer) are in Phase I trials Preliminary findings support the hypoth-esis that 4-1BB-specific agonist activity will increase T cell and natural killer cell proliferation and activity68 A Phase II trial of urelumab in patients with melanoma was terminated early owing to a high incidence of grade 4 hepatotoxicity69 New trials with urelumab are under-way to establish a safe and efficacious dose includ-ing a monotherapy trial in patients with advanced or

Box 2 | Lessons learnt from CTLA4 inhibitors

In both preclinical and clinical models cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors were developed in combination with other anticancer therapies Anti-CTLA4 monotherapy was not effective in the poorly immunogenic B16 melanoma mouse model however the combination of a CTLA4 inhibitor with a granulocytendashmacrophage colony stimulating factor (GM-CSF)-producing tumour vaccine resulted in tumour rejection as well as immune-related vitiligo215 Surviving mice also rejected tumour re-challenge supporting the hypothesis that CTLA4 blockade can support the development of antitumour immune memory In clinical trials the combination of ipilimumab with the gp100 peptide vaccine did not improve the clinical benefit of ipilimumab6 Moreover in a Phase II trial of high-dose ipilimumab (10 mg per kg) and GM-CSF greater antitumour responses were observed and with less toxicity than with ipilimumab alone216 It is unclear whether GM-CSF is playing a protective role that limits toxicity Preclinical studies have shown that GM-CSF can recruit dendritic cells but also enhance regulatory T (T

Reg) cells

providing possible mechanisms for enhanced activity and reduced toxicity217 These results successfully informed the design of a Phase III trial

It is clear that development of anti-CTLA4 combination therapies have been limited by toxicity as seen in Phase I trials that combine CTLA4 inhibitors with small-molecule agents For example adding ipilimumab after 1 month of treatment with the BRAF inhibitor vemurafenib caused undue toxicity Eight of the first 10 patients enrolled developed asymptomatic grade 3 liver toxicity within 5 weeks of starting ipilimumab which was reversible with drug discontinuation or active suppression of the immune response with glucocorticoid steroids47 Similarly the dose-limiting toxicity observed in the combination trial of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor sunitinib with the CTLA4-blocking antibody tremelimumab was rapid-onset acute kidney injury that was reversible with steroids218 Both trials were terminated due to toxicity Separate from its toxicity profile mechanistically anti-CTLA4 therapy may not be an optimal backbone for improving efficacy with other therapies When ipilimumab was combined with high-dose IL-2 ipilimumab failed to show any synergy producing only a 22 response rate little more than either therapy alone219 Similarly the Phase III trial combining ipilimumab with the chemotherapy dacarbazine demonstrated that ipilimumab in combination with dacarbazine provided an improved survival benefit over dacarbazine alone but the survival rate was comparable with subsequent trials evaluating ipilimumab as monotherapy6220 Although some combinations may fail to synergize the antitumour effect others may even blunt the antitumour effect Therefore given the multitude of potential combinations rational design is essential

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Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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copy 2015 Macmillan Publishers Limited All rights reserved

antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 6: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

Table 1 | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

CTLA4 Ipilimumab FDA approved

Tremelimumab Phase III

PD1ndashPDL1 Pembrolizumab (PD1) FDA approved

Nivolumab (PD1) FDA approved

Atezolizumab (formerly MPDL3280A) (PDL1) Phase III

MEDI4736 (PDL1) Phase III

Avelumab (PDL1) Phase I

PDR001 (PD1) Phase I

TNF and TNFR superfamilies

4-1BBndash4-1BB ligand Urelumab PF-05082566 Phase II

OX40ndashOX40 ligand MEDI6469 Phase II

GITR TRX518 Phase I

CD27 Varlilumab Phase II

TNFRSF25ndashTL1A Preclinical

CD40ndashCD40 ligand CP-870893 Phase I

HVEMndashLIGHTndashLTA Preclinical

HVEMndashBTLAndashCD160 Preclinical

IgSF

LAG3 BMS-986016 Phase I

TIM3 Preclinical

Siglecs Preclinical

B7 and CD28‑related proteins

ICOSndashICOS ligand Preclinical

B7-H3 MGA271 Phase I

B7-H4 Preclinical

VISTA Preclinical

HHLA2ndashTMIGD2 Preclinical

Butyrophilins including BTNL2 Preclinical

CD244ndashCD48 Preclinical

TIGIT and PVR family members Preclinical

Natural killer cell targets

KIRs Lirilumab Phase II

ILTs and LIRs Preclinical

NKG2D and NKG2A IPH2201 Phase I

MICA and MICB Preclinical

CD244 Preclinical

Suppressive myeloid cells

CSF1R Emactuzumab Phase I

Soluble mediators

IDO INCB024360 Phase II

TGFβ Galunisertib Phase I

AdenosinendashCD39ndashCD73 Preclinical

CXCR4ndashCXCL12 Ulocuplumab BKT140 Phase III

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that PD1 or PDL1 blockade combined with BRAF inhibition increased the activity of tumour -infiltrating lymphocytes and prolonged survival46 Based on such observations clinical trials evaluating combinations of PD1 pathway inhibitors with BRAF inhibitors such as vemurafenib (NCT01656642) or dabrafenib (Tafinlar GlaxoSmithKline) and with MEK inhibi-tors such as trametinib (Mekinist GlaxoSmithKline) (NCT02224781) are now underway in melanoma Although a Phase I trial combining ipilimumab plus vemurafenib was terminated early owing to hepatotox-icity47 preliminary safety data from the combination trial of MEDI4736 (a PDL1-specific mAb developed by AstraZeneca) dabrafenib and trametinib appear promis-ing (n = 26) (NCT02027961)48

Combinations with VEGF-directed therapy The VEGFR tyrosine kinase inhibitor sunitinib blocks growth fac-tor signalling in tumour and vascular cells and also reduces the accumulation of MDSCs within tumours MDSCs are critical to the maintenance of the tumour microenvironment and are potent inhibitors of immune responses49 The impact of sunitinib on vascular cells and MDSCs is consistent with an impact on the tumour microenvironment a subject of substantial interest in rational immunotherapy design In studies of the effect of sunitinib on primary kidney cancer tumours 3 out of 8 kidney tumour specimens had low levels of MDSCs and normal T cell production of IFNγ whereas 5 out of 8 specimens showed high intratumoural MDSCs and low T cell production of IFNγ50 Such data have sup-ported the design of a Phase I trial combining nivolumab with either sunitinib or pazopanib as second-line ther-apy in patients with kidney cancer22 Notably response rates with the Phase I combinations were greater than expected for each agent individually22 Increased grade 3ndash4 toxicities observed with this combination were largely reversible by treatment with steroids or delaying or stopping further drug doses

Targeting VEGF directly may result in a more effec-tive combination than inhibiting the receptor Kidney cancers that progress on VEGFR tyrosine kinase inhibi-tor therapy are associated with increased VEGF produc-tion by the tumour51 Many tumour types also secrete VEGF and elevated VEGF serum levels are a marker of poor prognosis in diverse cancer indications52ndash54 VEGF can be immunosuppressive because its natural role is to support tissue remodelling and repair For example by removing signalling through VEGFR VEGF blockade can enhance dendritic cell function and subsequent T cell activation5556 Thus it is promising that the combination of the VEGF-specific mAb bevacizumab (Avastin GenentechRoche) and the PDL1-specific mAb atezolizumab (formerly known as MPDL3280A developed by GenentechRoche) has demonstrated only modest toxicity in Phase I trials57 Such observations pro-vide a rationale for why this combination therapy may be more effective in kidney cancer than monotherapy either as first-line therapy or after progression (when VEGF secretion is upregulated)

VEGF has many functions in addition to angio-genesis and can signal through receptors other than VEGF tyrosine kinase receptors For example neuro-pilins are multifunctional non-tyrosine kinase recep-tors that bind to specific isoforms of VEGF and to TGFβ and semaphorins58 Semaphorins are linked to tumour proliferation and survival in addition to their role in axonal guidance Neuropilin is also expressed on a subset of human TReg cells (FIG 3) and on a specialized subset of dendritic cells termed plasmacytoid dendritic cells although the functional relevance of this expres-sion in the immune system is unclear5960 Neuropilin is upregulated in multiple tumour types and expres-sion correlates with tumour progression61 A Phase I trial of MNRP1685A (a human immunoglobulin G1 (IgG1) mAb developed by Roche) which targets the VEGF-binding domain of neuropilin 1 demonstrated tolerability62 However a Phase Ib trial combining an

Other

Phosphatidylserine Bavituximab Phase IIIII

SIRPAndashCD47 CC-90002 Phase I

VEGF Bevacizumab FDA approved

Neuropilin MNRP1685A Phase I

BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CSF1R macrophage colony-stimulating factor receptor 1 CTLA4 cytotoxic T lymphocyte antigen 4 CXCL12 chemokine (C-X-C motif) ligand 12 CXCR4 C-X-C chemokine receptor type 4 GITR glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator IDO indoleamine 23-dioxygenase IgSF immunoglobulin superfamily ILT immunoglobulin-like transcript KIR killer inhibitory immunoglobulin-like receptor LAG3 lymphocyte activation gene 3 protein LIR leukocyte immunoglobulin-like LTA lymphotoxin-α MIC MHC class I polypeptide-related sequence PD1 programmed cell death protein 1 PDL1 programmed cell death 1 ligand 1 PVR poliovirus receptor SIRPA signal-regulatory protein- TGFβ transforming growth factor-β TIGIT T cell immunoreceptor with immunoglobulin and ITIM domains TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 TNFRSF25 TNFR superfamily member 25 TNFR TNF receptor VEGF vascular endothelial growth factor VISTA V-domain immunoglobulin suppressor of T cell activation Plerixafor (Mozobil GenzymeSanofi) is approved by the US Food and Drug Administration not as an antitumour therapy but as a bone marrow mobilizing agent for bone marrow transplantation

Table 1 (cont) | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

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TNF receptors(TNFRs) A large family of proteins associated with diverse cellular activities including the activation of lymphocytes

TNFR superfamily(TNFRSF) A group containing approximately 30 type I or type III membrane proteins and several secreted proteins TNFRSF receptors are characterized by the presence of one to four extracellular cysteine-rich ligand-binding domains

anti-neuropilin agent the VEGF-specific mAb beva-cizumab and chemotherapy resulted in greater than expected proteinuria63 This result suggests that dual blockade of both VEGF and neuropilin may be lim-ited by toxicity However the future development of a neuropilin-specific mAb may have a role independent of VEGF function by targeting the immune response through TReg cells The recent finding that semaphorin 4A signals through neuropilin 1 to regulate TReg cell function in the tumour environment suggests that tar-geting this pathway may deactivate TReg cells facilitat-ing the development of antitumour immunity64 (FIG 3) The role of neuropilin 1 on TReg cells is being intensively investigated with the hope that inhibiting this activity of neuropilin 1 can selectively target TReg cells

New targets the TNFR superfamilyConcurrent with the discovery and targeting of CD28 and CTLA4 members of the tumour necrosis factor (TNF) family of proteins were identified and developed as some of the first molecularly defined immunothera-peutics Local infusion of TNF and lymphotoxin-α has

been used as a treatment for primary melanoma and sarcoma for decades although with limited efficacy and pronounced toxicity65 Other early attempts targeted various TNF receptors (TNFRs) with agonist antibodies to directly induce tumour cell death Examples of this class of therapeutics include antibodies directed against TRAIL receptor 2 (also known as TNFRSF10B) lymphotoxin-β receptor (also known as TNFRSF3) and FN14 (also known as TNFRSF12A) Some of these approaches were limited by toxicity notably hepatic toxicity The next wave of antibody development target-ing this receptor family came with the identification of 4-1BB (also known as CD137TNFRSF9) and related TNFRs as critical regulators of previously activated T cells66 There are six receptors in the TNFR superfamily (TNFRSF) currently receiving attention as immune-activating agents these are discussed below In contrast to immune checkpoint targets such as PD1 the goal of most of these antibodies is to activate the receptors thus they act as agonists (FIG 1) The driving concept for com-bination therapy is that TNFRSF agonists will further activate and support the antitumour immune response released by immune checkpoint inhibition

4-1BB 4-1BB is a co-stimulatory receptor on T cells and natural killer cells and it is expressed on neutrophils and myeloid lineage cells including some subpopula-tions of dendritic cells Ligation of 4-1BB on T cells and natural killer cells induces cell activation survival and enhances effector functions 4-1BB is expressed rapidly after T cell activation and its expression continues for up to 48 hours 4-1BB is found on all activated T cell subsets including TReg cells The pleiotropic activity of 4-1BB agonist antibodies is apparent in preclinical studies in which the effects varied depending on the models Preclinical data demonstrated 4-1BB effects on CD8+ T cell proliferation and survival In some model systems effects on CD4+ T cells have been reported The role of 4-1BB in TReg cell function is confusing as conflicting data suggest that 4-1BB activity can either expand or restrict TReg cell activation Importantly 41bb knockout mice can develop autoimmunity suggesting a role for 4-1BB in maintaining immune homeostasis67 Furthermore 41bb knockout mice are deficient in their ability to reject tumours in tumour challenge mod-els demonstrating that effective antitumour memory requires 4-1BB Some 4-1BB-deficient mice spontane-ously develop B cell lymphomas as they age but whether this is a consequence of underlying proliferation of auto-immune B cells or defective immunoediting is unclear

Two agonistic 4-1BB-specific antibodies urelumab (an IgG4 mAb developed by Bristol-Meyers Squibb) and PF-05082566 (an IgG2 mAb developed by Pfizer) are in Phase I trials Preliminary findings support the hypoth-esis that 4-1BB-specific agonist activity will increase T cell and natural killer cell proliferation and activity68 A Phase II trial of urelumab in patients with melanoma was terminated early owing to a high incidence of grade 4 hepatotoxicity69 New trials with urelumab are under-way to establish a safe and efficacious dose includ-ing a monotherapy trial in patients with advanced or

Box 2 | Lessons learnt from CTLA4 inhibitors

In both preclinical and clinical models cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors were developed in combination with other anticancer therapies Anti-CTLA4 monotherapy was not effective in the poorly immunogenic B16 melanoma mouse model however the combination of a CTLA4 inhibitor with a granulocytendashmacrophage colony stimulating factor (GM-CSF)-producing tumour vaccine resulted in tumour rejection as well as immune-related vitiligo215 Surviving mice also rejected tumour re-challenge supporting the hypothesis that CTLA4 blockade can support the development of antitumour immune memory In clinical trials the combination of ipilimumab with the gp100 peptide vaccine did not improve the clinical benefit of ipilimumab6 Moreover in a Phase II trial of high-dose ipilimumab (10 mg per kg) and GM-CSF greater antitumour responses were observed and with less toxicity than with ipilimumab alone216 It is unclear whether GM-CSF is playing a protective role that limits toxicity Preclinical studies have shown that GM-CSF can recruit dendritic cells but also enhance regulatory T (T

Reg) cells

providing possible mechanisms for enhanced activity and reduced toxicity217 These results successfully informed the design of a Phase III trial

It is clear that development of anti-CTLA4 combination therapies have been limited by toxicity as seen in Phase I trials that combine CTLA4 inhibitors with small-molecule agents For example adding ipilimumab after 1 month of treatment with the BRAF inhibitor vemurafenib caused undue toxicity Eight of the first 10 patients enrolled developed asymptomatic grade 3 liver toxicity within 5 weeks of starting ipilimumab which was reversible with drug discontinuation or active suppression of the immune response with glucocorticoid steroids47 Similarly the dose-limiting toxicity observed in the combination trial of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor sunitinib with the CTLA4-blocking antibody tremelimumab was rapid-onset acute kidney injury that was reversible with steroids218 Both trials were terminated due to toxicity Separate from its toxicity profile mechanistically anti-CTLA4 therapy may not be an optimal backbone for improving efficacy with other therapies When ipilimumab was combined with high-dose IL-2 ipilimumab failed to show any synergy producing only a 22 response rate little more than either therapy alone219 Similarly the Phase III trial combining ipilimumab with the chemotherapy dacarbazine demonstrated that ipilimumab in combination with dacarbazine provided an improved survival benefit over dacarbazine alone but the survival rate was comparable with subsequent trials evaluating ipilimumab as monotherapy6220 Although some combinations may fail to synergize the antitumour effect others may even blunt the antitumour effect Therefore given the multitude of potential combinations rational design is essential

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Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

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predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 7: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

that PD1 or PDL1 blockade combined with BRAF inhibition increased the activity of tumour -infiltrating lymphocytes and prolonged survival46 Based on such observations clinical trials evaluating combinations of PD1 pathway inhibitors with BRAF inhibitors such as vemurafenib (NCT01656642) or dabrafenib (Tafinlar GlaxoSmithKline) and with MEK inhibi-tors such as trametinib (Mekinist GlaxoSmithKline) (NCT02224781) are now underway in melanoma Although a Phase I trial combining ipilimumab plus vemurafenib was terminated early owing to hepatotox-icity47 preliminary safety data from the combination trial of MEDI4736 (a PDL1-specific mAb developed by AstraZeneca) dabrafenib and trametinib appear promis-ing (n = 26) (NCT02027961)48

Combinations with VEGF-directed therapy The VEGFR tyrosine kinase inhibitor sunitinib blocks growth fac-tor signalling in tumour and vascular cells and also reduces the accumulation of MDSCs within tumours MDSCs are critical to the maintenance of the tumour microenvironment and are potent inhibitors of immune responses49 The impact of sunitinib on vascular cells and MDSCs is consistent with an impact on the tumour microenvironment a subject of substantial interest in rational immunotherapy design In studies of the effect of sunitinib on primary kidney cancer tumours 3 out of 8 kidney tumour specimens had low levels of MDSCs and normal T cell production of IFNγ whereas 5 out of 8 specimens showed high intratumoural MDSCs and low T cell production of IFNγ50 Such data have sup-ported the design of a Phase I trial combining nivolumab with either sunitinib or pazopanib as second-line ther-apy in patients with kidney cancer22 Notably response rates with the Phase I combinations were greater than expected for each agent individually22 Increased grade 3ndash4 toxicities observed with this combination were largely reversible by treatment with steroids or delaying or stopping further drug doses

Targeting VEGF directly may result in a more effec-tive combination than inhibiting the receptor Kidney cancers that progress on VEGFR tyrosine kinase inhibi-tor therapy are associated with increased VEGF produc-tion by the tumour51 Many tumour types also secrete VEGF and elevated VEGF serum levels are a marker of poor prognosis in diverse cancer indications52ndash54 VEGF can be immunosuppressive because its natural role is to support tissue remodelling and repair For example by removing signalling through VEGFR VEGF blockade can enhance dendritic cell function and subsequent T cell activation5556 Thus it is promising that the combination of the VEGF-specific mAb bevacizumab (Avastin GenentechRoche) and the PDL1-specific mAb atezolizumab (formerly known as MPDL3280A developed by GenentechRoche) has demonstrated only modest toxicity in Phase I trials57 Such observations pro-vide a rationale for why this combination therapy may be more effective in kidney cancer than monotherapy either as first-line therapy or after progression (when VEGF secretion is upregulated)

VEGF has many functions in addition to angio-genesis and can signal through receptors other than VEGF tyrosine kinase receptors For example neuro-pilins are multifunctional non-tyrosine kinase recep-tors that bind to specific isoforms of VEGF and to TGFβ and semaphorins58 Semaphorins are linked to tumour proliferation and survival in addition to their role in axonal guidance Neuropilin is also expressed on a subset of human TReg cells (FIG 3) and on a specialized subset of dendritic cells termed plasmacytoid dendritic cells although the functional relevance of this expres-sion in the immune system is unclear5960 Neuropilin is upregulated in multiple tumour types and expres-sion correlates with tumour progression61 A Phase I trial of MNRP1685A (a human immunoglobulin G1 (IgG1) mAb developed by Roche) which targets the VEGF-binding domain of neuropilin 1 demonstrated tolerability62 However a Phase Ib trial combining an

Other

Phosphatidylserine Bavituximab Phase IIIII

SIRPAndashCD47 CC-90002 Phase I

VEGF Bevacizumab FDA approved

Neuropilin MNRP1685A Phase I

BTLA B and T lymphocyte attenuator BTNL2 butyrophilin-like protein 2 CSF1R macrophage colony-stimulating factor receptor 1 CTLA4 cytotoxic T lymphocyte antigen 4 CXCL12 chemokine (C-X-C motif) ligand 12 CXCR4 C-X-C chemokine receptor type 4 GITR glucocorticoid-induced tumour necrosis factor receptor (TNFR)-related protein HHLA2 HERV-H LTR-associating protein 2 HVEM herpes virus entry mediator ICOS inducible T cell co-stimulator IDO indoleamine 23-dioxygenase IgSF immunoglobulin superfamily ILT immunoglobulin-like transcript KIR killer inhibitory immunoglobulin-like receptor LAG3 lymphocyte activation gene 3 protein LIR leukocyte immunoglobulin-like LTA lymphotoxin-α MIC MHC class I polypeptide-related sequence PD1 programmed cell death protein 1 PDL1 programmed cell death 1 ligand 1 PVR poliovirus receptor SIRPA signal-regulatory protein- TGFβ transforming growth factor-β TIGIT T cell immunoreceptor with immunoglobulin and ITIM domains TIM3 T cell immunoglobulin mucin 3 TL1A TNF-like ligand 1A TMIGD2 transmembrane and immunoglobulin domain-containing protein 2 TNFRSF25 TNFR superfamily member 25 TNFR TNF receptor VEGF vascular endothelial growth factor VISTA V-domain immunoglobulin suppressor of T cell activation Plerixafor (Mozobil GenzymeSanofi) is approved by the US Food and Drug Administration not as an antitumour therapy but as a bone marrow mobilizing agent for bone marrow transplantation

Table 1 (cont) | Immunological targets currently in clinical or preclinical development

Immunological pathway Examples in clinical trials Most advanced stage of clinical development

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 567

copy 2015 Macmillan Publishers Limited All rights reserved

TNF receptors(TNFRs) A large family of proteins associated with diverse cellular activities including the activation of lymphocytes

TNFR superfamily(TNFRSF) A group containing approximately 30 type I or type III membrane proteins and several secreted proteins TNFRSF receptors are characterized by the presence of one to four extracellular cysteine-rich ligand-binding domains

anti-neuropilin agent the VEGF-specific mAb beva-cizumab and chemotherapy resulted in greater than expected proteinuria63 This result suggests that dual blockade of both VEGF and neuropilin may be lim-ited by toxicity However the future development of a neuropilin-specific mAb may have a role independent of VEGF function by targeting the immune response through TReg cells The recent finding that semaphorin 4A signals through neuropilin 1 to regulate TReg cell function in the tumour environment suggests that tar-geting this pathway may deactivate TReg cells facilitat-ing the development of antitumour immunity64 (FIG 3) The role of neuropilin 1 on TReg cells is being intensively investigated with the hope that inhibiting this activity of neuropilin 1 can selectively target TReg cells

New targets the TNFR superfamilyConcurrent with the discovery and targeting of CD28 and CTLA4 members of the tumour necrosis factor (TNF) family of proteins were identified and developed as some of the first molecularly defined immunothera-peutics Local infusion of TNF and lymphotoxin-α has

been used as a treatment for primary melanoma and sarcoma for decades although with limited efficacy and pronounced toxicity65 Other early attempts targeted various TNF receptors (TNFRs) with agonist antibodies to directly induce tumour cell death Examples of this class of therapeutics include antibodies directed against TRAIL receptor 2 (also known as TNFRSF10B) lymphotoxin-β receptor (also known as TNFRSF3) and FN14 (also known as TNFRSF12A) Some of these approaches were limited by toxicity notably hepatic toxicity The next wave of antibody development target-ing this receptor family came with the identification of 4-1BB (also known as CD137TNFRSF9) and related TNFRs as critical regulators of previously activated T cells66 There are six receptors in the TNFR superfamily (TNFRSF) currently receiving attention as immune-activating agents these are discussed below In contrast to immune checkpoint targets such as PD1 the goal of most of these antibodies is to activate the receptors thus they act as agonists (FIG 1) The driving concept for com-bination therapy is that TNFRSF agonists will further activate and support the antitumour immune response released by immune checkpoint inhibition

4-1BB 4-1BB is a co-stimulatory receptor on T cells and natural killer cells and it is expressed on neutrophils and myeloid lineage cells including some subpopula-tions of dendritic cells Ligation of 4-1BB on T cells and natural killer cells induces cell activation survival and enhances effector functions 4-1BB is expressed rapidly after T cell activation and its expression continues for up to 48 hours 4-1BB is found on all activated T cell subsets including TReg cells The pleiotropic activity of 4-1BB agonist antibodies is apparent in preclinical studies in which the effects varied depending on the models Preclinical data demonstrated 4-1BB effects on CD8+ T cell proliferation and survival In some model systems effects on CD4+ T cells have been reported The role of 4-1BB in TReg cell function is confusing as conflicting data suggest that 4-1BB activity can either expand or restrict TReg cell activation Importantly 41bb knockout mice can develop autoimmunity suggesting a role for 4-1BB in maintaining immune homeostasis67 Furthermore 41bb knockout mice are deficient in their ability to reject tumours in tumour challenge mod-els demonstrating that effective antitumour memory requires 4-1BB Some 4-1BB-deficient mice spontane-ously develop B cell lymphomas as they age but whether this is a consequence of underlying proliferation of auto-immune B cells or defective immunoediting is unclear

Two agonistic 4-1BB-specific antibodies urelumab (an IgG4 mAb developed by Bristol-Meyers Squibb) and PF-05082566 (an IgG2 mAb developed by Pfizer) are in Phase I trials Preliminary findings support the hypoth-esis that 4-1BB-specific agonist activity will increase T cell and natural killer cell proliferation and activity68 A Phase II trial of urelumab in patients with melanoma was terminated early owing to a high incidence of grade 4 hepatotoxicity69 New trials with urelumab are under-way to establish a safe and efficacious dose includ-ing a monotherapy trial in patients with advanced or

Box 2 | Lessons learnt from CTLA4 inhibitors

In both preclinical and clinical models cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors were developed in combination with other anticancer therapies Anti-CTLA4 monotherapy was not effective in the poorly immunogenic B16 melanoma mouse model however the combination of a CTLA4 inhibitor with a granulocytendashmacrophage colony stimulating factor (GM-CSF)-producing tumour vaccine resulted in tumour rejection as well as immune-related vitiligo215 Surviving mice also rejected tumour re-challenge supporting the hypothesis that CTLA4 blockade can support the development of antitumour immune memory In clinical trials the combination of ipilimumab with the gp100 peptide vaccine did not improve the clinical benefit of ipilimumab6 Moreover in a Phase II trial of high-dose ipilimumab (10 mg per kg) and GM-CSF greater antitumour responses were observed and with less toxicity than with ipilimumab alone216 It is unclear whether GM-CSF is playing a protective role that limits toxicity Preclinical studies have shown that GM-CSF can recruit dendritic cells but also enhance regulatory T (T

Reg) cells

providing possible mechanisms for enhanced activity and reduced toxicity217 These results successfully informed the design of a Phase III trial

It is clear that development of anti-CTLA4 combination therapies have been limited by toxicity as seen in Phase I trials that combine CTLA4 inhibitors with small-molecule agents For example adding ipilimumab after 1 month of treatment with the BRAF inhibitor vemurafenib caused undue toxicity Eight of the first 10 patients enrolled developed asymptomatic grade 3 liver toxicity within 5 weeks of starting ipilimumab which was reversible with drug discontinuation or active suppression of the immune response with glucocorticoid steroids47 Similarly the dose-limiting toxicity observed in the combination trial of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor sunitinib with the CTLA4-blocking antibody tremelimumab was rapid-onset acute kidney injury that was reversible with steroids218 Both trials were terminated due to toxicity Separate from its toxicity profile mechanistically anti-CTLA4 therapy may not be an optimal backbone for improving efficacy with other therapies When ipilimumab was combined with high-dose IL-2 ipilimumab failed to show any synergy producing only a 22 response rate little more than either therapy alone219 Similarly the Phase III trial combining ipilimumab with the chemotherapy dacarbazine demonstrated that ipilimumab in combination with dacarbazine provided an improved survival benefit over dacarbazine alone but the survival rate was comparable with subsequent trials evaluating ipilimumab as monotherapy6220 Although some combinations may fail to synergize the antitumour effect others may even blunt the antitumour effect Therefore given the multitude of potential combinations rational design is essential

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Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

580 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 8: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

TNF receptors(TNFRs) A large family of proteins associated with diverse cellular activities including the activation of lymphocytes

TNFR superfamily(TNFRSF) A group containing approximately 30 type I or type III membrane proteins and several secreted proteins TNFRSF receptors are characterized by the presence of one to four extracellular cysteine-rich ligand-binding domains

anti-neuropilin agent the VEGF-specific mAb beva-cizumab and chemotherapy resulted in greater than expected proteinuria63 This result suggests that dual blockade of both VEGF and neuropilin may be lim-ited by toxicity However the future development of a neuropilin-specific mAb may have a role independent of VEGF function by targeting the immune response through TReg cells The recent finding that semaphorin 4A signals through neuropilin 1 to regulate TReg cell function in the tumour environment suggests that tar-geting this pathway may deactivate TReg cells facilitat-ing the development of antitumour immunity64 (FIG 3) The role of neuropilin 1 on TReg cells is being intensively investigated with the hope that inhibiting this activity of neuropilin 1 can selectively target TReg cells

New targets the TNFR superfamilyConcurrent with the discovery and targeting of CD28 and CTLA4 members of the tumour necrosis factor (TNF) family of proteins were identified and developed as some of the first molecularly defined immunothera-peutics Local infusion of TNF and lymphotoxin-α has

been used as a treatment for primary melanoma and sarcoma for decades although with limited efficacy and pronounced toxicity65 Other early attempts targeted various TNF receptors (TNFRs) with agonist antibodies to directly induce tumour cell death Examples of this class of therapeutics include antibodies directed against TRAIL receptor 2 (also known as TNFRSF10B) lymphotoxin-β receptor (also known as TNFRSF3) and FN14 (also known as TNFRSF12A) Some of these approaches were limited by toxicity notably hepatic toxicity The next wave of antibody development target-ing this receptor family came with the identification of 4-1BB (also known as CD137TNFRSF9) and related TNFRs as critical regulators of previously activated T cells66 There are six receptors in the TNFR superfamily (TNFRSF) currently receiving attention as immune-activating agents these are discussed below In contrast to immune checkpoint targets such as PD1 the goal of most of these antibodies is to activate the receptors thus they act as agonists (FIG 1) The driving concept for com-bination therapy is that TNFRSF agonists will further activate and support the antitumour immune response released by immune checkpoint inhibition

4-1BB 4-1BB is a co-stimulatory receptor on T cells and natural killer cells and it is expressed on neutrophils and myeloid lineage cells including some subpopula-tions of dendritic cells Ligation of 4-1BB on T cells and natural killer cells induces cell activation survival and enhances effector functions 4-1BB is expressed rapidly after T cell activation and its expression continues for up to 48 hours 4-1BB is found on all activated T cell subsets including TReg cells The pleiotropic activity of 4-1BB agonist antibodies is apparent in preclinical studies in which the effects varied depending on the models Preclinical data demonstrated 4-1BB effects on CD8+ T cell proliferation and survival In some model systems effects on CD4+ T cells have been reported The role of 4-1BB in TReg cell function is confusing as conflicting data suggest that 4-1BB activity can either expand or restrict TReg cell activation Importantly 41bb knockout mice can develop autoimmunity suggesting a role for 4-1BB in maintaining immune homeostasis67 Furthermore 41bb knockout mice are deficient in their ability to reject tumours in tumour challenge mod-els demonstrating that effective antitumour memory requires 4-1BB Some 4-1BB-deficient mice spontane-ously develop B cell lymphomas as they age but whether this is a consequence of underlying proliferation of auto-immune B cells or defective immunoediting is unclear

Two agonistic 4-1BB-specific antibodies urelumab (an IgG4 mAb developed by Bristol-Meyers Squibb) and PF-05082566 (an IgG2 mAb developed by Pfizer) are in Phase I trials Preliminary findings support the hypoth-esis that 4-1BB-specific agonist activity will increase T cell and natural killer cell proliferation and activity68 A Phase II trial of urelumab in patients with melanoma was terminated early owing to a high incidence of grade 4 hepatotoxicity69 New trials with urelumab are under-way to establish a safe and efficacious dose includ-ing a monotherapy trial in patients with advanced or

Box 2 | Lessons learnt from CTLA4 inhibitors

In both preclinical and clinical models cytotoxic T lymphocyte antigen 4 (CTLA4) inhibitors were developed in combination with other anticancer therapies Anti-CTLA4 monotherapy was not effective in the poorly immunogenic B16 melanoma mouse model however the combination of a CTLA4 inhibitor with a granulocytendashmacrophage colony stimulating factor (GM-CSF)-producing tumour vaccine resulted in tumour rejection as well as immune-related vitiligo215 Surviving mice also rejected tumour re-challenge supporting the hypothesis that CTLA4 blockade can support the development of antitumour immune memory In clinical trials the combination of ipilimumab with the gp100 peptide vaccine did not improve the clinical benefit of ipilimumab6 Moreover in a Phase II trial of high-dose ipilimumab (10 mg per kg) and GM-CSF greater antitumour responses were observed and with less toxicity than with ipilimumab alone216 It is unclear whether GM-CSF is playing a protective role that limits toxicity Preclinical studies have shown that GM-CSF can recruit dendritic cells but also enhance regulatory T (T

Reg) cells

providing possible mechanisms for enhanced activity and reduced toxicity217 These results successfully informed the design of a Phase III trial

It is clear that development of anti-CTLA4 combination therapies have been limited by toxicity as seen in Phase I trials that combine CTLA4 inhibitors with small-molecule agents For example adding ipilimumab after 1 month of treatment with the BRAF inhibitor vemurafenib caused undue toxicity Eight of the first 10 patients enrolled developed asymptomatic grade 3 liver toxicity within 5 weeks of starting ipilimumab which was reversible with drug discontinuation or active suppression of the immune response with glucocorticoid steroids47 Similarly the dose-limiting toxicity observed in the combination trial of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor sunitinib with the CTLA4-blocking antibody tremelimumab was rapid-onset acute kidney injury that was reversible with steroids218 Both trials were terminated due to toxicity Separate from its toxicity profile mechanistically anti-CTLA4 therapy may not be an optimal backbone for improving efficacy with other therapies When ipilimumab was combined with high-dose IL-2 ipilimumab failed to show any synergy producing only a 22 response rate little more than either therapy alone219 Similarly the Phase III trial combining ipilimumab with the chemotherapy dacarbazine demonstrated that ipilimumab in combination with dacarbazine provided an improved survival benefit over dacarbazine alone but the survival rate was comparable with subsequent trials evaluating ipilimumab as monotherapy6220 Although some combinations may fail to synergize the antitumour effect others may even blunt the antitumour effect Therefore given the multitude of potential combinations rational design is essential

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Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

580 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

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copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 9: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

Antibody-dependent cell-mediated cytotoxicity(ADCC) Cell killing mediated by natural killer cells macrophages neutrophils and eosinophils through antibodies bound to Fc receptors expressed by these effector cells

Natural killer T cellsA type of lymphocyte that is thought to have the functions of both activated T cells and natural killer cells

T cell memoryThe process of developing long-lived and self-renewing T cells that retain specificity for an antigen after the antigen has been removed allowing rapid reactivation of the immune response

Fc receptorA type of cell surface receptor that binds to the crystallizable fragment (Fc) region of antibodies to trigger a variety of cellular responses

metastatic solid tumours or with relapsed or refractory non-Hodgkin lymphoma (NCT01471210) In addition to a combination trial with urelumab plus rituximab for non-Hodgkin lymphoma (NCT01775631) the following combination trials are ongoing elotuzumab (a signalling lymphocyte activation molecule (SLAM) family member 7 (SLAMF7)-specific mAb developed by Bristol-Myers SquibbAbbVie) with urelumab or lirilumab (a killer inhibitory immunoglobulin-like receptor (KIR)-specific mAb developed by Bristol-Myers SquibbInnate Pharma) for multiple myeloma (NCT02252263) urelumab plus nivolumab in solid tumours and in non-Hodgkin lym-phoma (NCT02253992) and urelumab plus cetuximab (Erbitux an EGFR-specific mAb Bristol-Myers SquibbEli Lilly and CompanyMerck Serono) in colorectal can-cer and in head and neck carcinoma (NCT02110082)

PF-05082566 has been evaluated in a dose -escalation clinical study in mixed solid tumours and in non-Hodgkin lymphoma Clinical efficacy was observed in 9 out of 24 evaluable patients and notably toxicity was generally mild70 A Phase I trial of the combination of PF-05082566 plus rituximab in non-Hodgkin lym-phoma (NCT01307267) reported efficacy in rituximab-refractory non-Hodgkin lymphoma and no grade 3 toxicity71 Ongoing studies include monotherapy expan-sion cohorts in melanoma and Merkel cell carcinoma and combination trials with pembrolizumab in solid tumours (NCT02179918) An interesting recent study showed that tumour-depleting antibody therapies such as cetuximab can upregulate 4-1BB on natural killer cells This provides a clear rationale for the combination of 4-1BB-specific agonist antibody therapy with tumour-depleting antibodies which function through antibody-dependent cell-mediated cytotoxicity (ADCC) that can be mediated by natural killer cells72

4-1BB expression appears to accurately identify tumour-reactive tumour-infiltrating lymphocytes in some tumour types such as ovarian cancer73 and therefore may serve as a biomarker for 4-1BB-specific therapeutics and other immune-targeting therapies The impact of agonist 4-1BB-specific antibodies on TReg cell function will also be important to understand clinically as activation of TReg cell-mediated suppression could negate any antitumour efficacy (FIG 3) With significant resources invested in ongoing clinical investigation of 4-1BB agonist mAbs we anticipate rapid advances in our understanding of the role of this pathway in immunotherapy

OX40 OX40 (also known as CD134TNFRSF4) is expressed on diverse T cell subsets natural killer cells natural killer T cells and neutrophils whereas its ligand OX40L (also known as CD252TNFSF4) is found on APCs including dendritic cells B cells and mac-rophages and more broadly during inflammation for example on activated endothelium This expression pat-tern suggests that the OX40LndashOX40 pathway supports the immune response during T cell activation OX40 is transiently expressed on activated T cells after T cell receptor engagement generally appearing after 12 hours and waning by day 4 Available data support a model

whereby OX40 activity supports the survival and expan-sion of activated T cell subsets and the establishment of T cell memory66 OX40 agonist antibody treatment can reactivate the memory T cell population66 Additional data suggest that OX40 engagement deactivates the TReg cell population within tumours which would fur-ther sustain effector T cell function In some murine models an agonist OX40-specific antibody can deplete TReg cells This activity is antibody specific and requires Fc receptor-mediated ADCC activation74

Clinical trials using OX40-specific agonist antibody were spurred on by impressive results in mouse tumour models as monotherapy and in combination with chem-otherapy irradiation targeted small-molecule therapeu-tics cytokines and adjuvants (for example IL-2 IL-12 and CpG) and other immunomodulatory antibodies (for example antibodies targeting 4-1BB PD1 TIM3 CTLA4 or TGFβ)74ndash76 The concept of OX40 plus 4-1BB agonist therapy for the treatment of solid tumours has also gained significant preclinical rationale The under-lying hypothesis is that the combination will better engage diverse immune cell types in the antitumour immune response The combination of OX40-specific and 4-1BB-specific agonist antibodies generates T cell responses including CD8+ cells that secrete very high lev-els of IFNγ and granzyme B without activation of the TReg cell subset77 The combination of PD1-specific antagonist antibody plus 4-1BB-specific agonist antibody provided T cell -mediated protection in diverse syngeneic tumour models78 Other examples from the preclinical literature include triple combinations of OX40 agonists with anti-bodies targeting PD1 CTLA4 and TIM3

There are several OX40 agonist antibodies in clini-cal development Results from a prostate cancer trial of a mouse IgG1 anti-human OX40 mAb (9B12 devel-oped by AgonOx) in combination with standard of care showed an increase in the number of circulating CD4+ T cells CD8+ T cells and natural killer cells support-ing the hypothesis that OX40 agonism promotes the proliferation and survival of activated T cells7980 There was no consistent expansion of the TReg cell subset and clinical responses of stable disease were observed As T cells must first be activated to express OX40 mono-therapy may not be the best setting for testing anti-OX40 agents Additional clinical development is proceed-ing with humanized or fully human mAbs including the OX40-specific antibody MEDI6469 (developed by AstraZeneca) in combination with the CTLA4-specific antibody tremelimumab (a fully human IgG2 mAb developed by Pfizer) the PDL1-specific antibody MEDI4736 (developed by AstraZeneca) and other thera-peutics such as rituximab (NCT02205333)

GITR The glucocorticoid-induced TNFR-related pro-tein (GITR also known as CD357TNFRSF18) is con-stitutively expressed on TReg cells GITR expression is upregulated on resting CD4+ and CD8+ T cells 24 hours after stimulation and this higher expression is main-tained for several days Other cells that can express GITR include dendritic cells monocytes and natural killer cells The GITR ligand (also known as TNFSF18) is highly

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expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

580 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 10: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

expressed on activated APCs and endothelial cells at sites of inflammation GITR appears to have a critical role in supporting T effector cell activity by inducing T cell pro-liferation and effector functions and by supporting T cell survival An indirect effect on the T effector cell compart-ment is GITR-mediated suppression of TReg cell activity The relative importance of these effects on different T cell populations as well as on natural killer cells is the subject of much discussion

In preclinical models a GITR agonist antibody produced impressive antitumour efficacy and was shown to synergize with anti-PD1 therapy to eliminate estab-lished tumours8182 Although the clinical development of GITR-specific antibodies is limited to date antibodies from GITR Inc Merck Agenus and others are in pre-clinical and early clinical development Looking ahead if the mechanism of action underlying anti-GITR activ-ity in tumour models includes downregulation of TReg cell activity then this pathway may be a good choice for combination therapy with T cell-activating therapeutics including other agonist TNFR-specific antibodies and the immune checkpoint inhibitors which can affect both T effector cells and TReg cells (FIGS 13)

CD27 The co-stimulatory molecule CD27 is constitu-tively expressed on most effector T cells memory B cells and a subset of natural killer cells CD27 is important for sustained T cell effector functions and the development of T cell memory By driving CD4+ T cell activity CD27 sup-ports germinal centre formation and B cell maturation and production of affinity-matured IgG antibodies CD27 is a marker of memory T cells highlighting the importance of this pathway in supporting T cell persistence CD27 expression on TReg cells is variable Finally CD27 drives the cytolytic activity of some subsets of natural killer cells66 Surprisingly CD70 the ligand for CD27 is expressed at very high levels on some tumours (for example renal cell carcinoma)83 This expression pattern is puzzling given the preclinical data supporting the relevance of CD27 in controlling tumour growth However renal cell carcinoma and other solid tumours express high levels of PDL1 and this immune evasion strategy may override any impact of CD70 and CD27 on the antitumour immune response

Ongoing clinical trials include a combination trial of the PD1-specific antibody nivolumab with the CD27-specific agonist antibody CDX-1127 (also known as varlilumab developed by Celldex) Early data from a Phase I dose-escalation study of CDX-1127 in non-Hodgkin lymphoma and solid tumours (NCT01460134) demonstrated tolerability and measureable clinical responses8485 There is strong interest in this pathway within the biopharmaceutical industry and new pro-grammes are anticipated to emerge

TNFRSF25 A newly recognized immune-activating receptor is TNFRSF25 (also known as DR3) its ligand is TNF-like ligand 1A (TL1A also known as TNFSF15) which is related to TNF-related weak inducer of apoptosis (TWEAK also known as TNFSF12)8687 The importance of the TL1AndashTNFRSF25 pathway was demonstrated in Tnfrsf25-deficient mice88 Strikingly the T cell expres-sion of TNFRSF25 was required for local T cell accu-mulation cytokine production and immunopathology in diverse inflammatory and autoimmune disease mod-els Furthermore it was demonstrated that expression of TL1A on mouse tumour cells triggers elimination of the tumour by CD8+ T cells and induces T cell memory such that mice are resistant to subsequent tumour chal-lenge89 Agonist TNFRSF25-specific antibody treatment was shown to preferentially support de novo T effector

Box 3 | Considerations in the design of immunomodulatory antibodies

Many of the therapeutic agents discussed here are monoclonal antibodies (mAbs) They have three proposed mechanisms of action depleting specific cell populations blocking inhibitory signals (antagonists) or engaging stimulatory signals (agonists) The specificity of these effects depends on the antibody variable regions that confer recognition of specific antigens but their functional effects crucially depend on the constant regions of the antibody that is the crystallizable fragment (Fc) domain (reviewed in REF 221) Many of the antibodies currently used in immunochemotherapy regimens such as rituximab or trastuzumab recognize tumour-restricted cell-surface proteins and kill the tumour cells via antibody-dependent cell-mediated cytotoxicity (ADCC) through signalling by their Fc domain through Fc receptors on effector cells By contrast immune checkpoint inhibitors are antibodies that aim to block the inhibitory signal on lymphocytes that is not deplete these cells but allow them to expand

Although pharmaceutical developers are keenly aware of the importance of the Fc region academic scientists are often not precise in their interpretation of the mechanism of action of an antibody in a mouse All too often any mAb that binds to a target is referred to as blocking after characterization with just a binding assay but whether the mechanism of action in vivo is ligand blockade or depletion of a particular cell population is not determined If the ligand-blocking capacity is not known the term targeting should be used rather than blocking

As human immunoglobulin G4 (IgG4) minimally engages the Fc receptor this isotype is frequently used in pharmaceutical development when blocking activity is desired without cell-depleting or agonist activity Hence most clinical programmed cell death protein 1 (PD1)-specific and programmed cell death 1 ligand 1 (PDL1)-specific mAbs are human IgG4 Human IgG1 engages the Fc receptor and complement and is therefore used when the desired mechanism of action is agonism or cell depletion The macrophage colony-stimulating factor receptor 1 (CSF1R)-specific therapeutic emactuzumab has an IgG1 Fc domain and was well tolerated in Phase I trials222 Additionally on-treatment biopsies supported the proof of the principle that immunosuppressive tumour-associated macrophages are depleted in tumours of treated patients222 Agonistic activity depends on the multimerization of the mAb on the surface of the Fc receptor-expressing cell facilitating cross linking of the antigen on the surface of the target cell Higher target antigen density appears to favour cell killing over agonism although specific protein families have their own unique rules of engagement Fortuitously the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab was designed as a human IgG1 before Fc functions were more thoroughly understood Although ipilimumab blocks the interactions between CTLA4 and CD80 or CD86 its mechanism of action is now thought to be ADCC depletion of tumour-infiltrating regulatory T cells that express high levels of cell surface CTLA4 (REFS 223224) An additional surprise is that the in vivo mechanism of action of OX40-specific mAbs depends on their cell killing activity rather than agonist activity74 Clearly there will continue to be surprises in the mechanism of action of therapeutic mAbs

Ideally mouse model experiments would use recombinant mAbs with mouse Fc domains to test blocking agonism or cell-depleting mechanisms of action However the nomenclature of antibody isotypes in mouse rat and human is not synonymous as the names were arbitrarily assigned based on electrophoretic gel mobilities Mouse IgG2a is considered the closest functional equivalent of human IgG1 Mouse IgG1 is considered the closest functional equivalent of human IgG4 Additional mutations such as D265A can further reduce the affinity of mouse IgG1 for the Fc receptor224 With the advanced state of antibody engineering technology the mAbs used in mouse models will hopefully improve to better model clinical use in humans

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Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

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predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 11: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

Immunoglobulin superfamilyA large group of proteins containing a structural domain related to the immunoglobulin (Ig) domain of antibodies Ig domains contain 70ndash110 amino acids with a charcteristic Ig fold giving them a sandwich-like structure composed of two sheets of anti-parallel β-strands

cell responses while maintaining TReg cell responses to self antigen90 The agonist TNFRSF25-specific antibody selec-tively expanded memory CD8+ T cells in a re-challenge setting more effectively than an agonist OX40-specific antibody91 This apparent function of TNFRSF25 in maintaining homeostasis may be a consequence of the role of this pathway in regulating gut immunity Although TNFSFR25 is chromosomally colocalized with 4-1BB OX40 GITR and CD27 sequence analysis sug-gests that TNFSFR25 is relatively divergent or perhaps

ancestral92 Thus we might expect that therapeutics directed against this pathway will have unique activity Agonist TNFRSF25-specific antibodies are in develop-ment (for example PTX-25 by Pelican Therapeutics)

CD40 In contrast to most of the targets discussed above which are mostly expressed on T cells and in some cases natural killer cells the TNFRSF member CD40 (also known as TNFRSF5) is constitutively expressed on APCs and diverse other cell types notably B cells The ligand for CD40 CD40L (also known as CD154TNFSF5) is expressed on activated T cells platelets and several other cell types CD40 activation promotes the matura-tion of APC function and expression of co-stimulatory molecules to increase the activation of antigen-specific T cells Preclinical programmes include agonist antibod-ies from Alligator Biosciences and several other compa-nies A Phase I trial combining the CD40-specific agonist CP-870893 (developed by Pfizer) and the CTLA4-specific antibody tremelimumab (NCT01103635) found colitis hypophysitis and uveitis to be the dose-limiting toxici-ties93 The most common treatment-related toxicity was grade 1ndash2 cytokine release syndrome which occurred within 24 hours of administration of the CD40-specific agonist in 792 patients with metastatic melanoma (n = 22) the objective response rate was 273 In mouse models CD40 agonistic treatment induced PDL1 upregulation on tumour-infiltrating monocytes and macrophages94 Combining CP-870893 with PD1- or PDL1-specific antibodies may be more effective but it also warrants careful dose-finding trials given the risk of hepatotoxicity seen in preclinical models95

An atypical TNFRSF HVEM HVEM (also known as TNFRSF14) originally identified as a herpes virus entry mediator is a TNFRSF member that can signal to inhibit immune responses in the context of antitu-mour responses HVEM is widely expressed on APCs endothelium and lymphocytes with the highest expres-sion levels detected on naive T cells HVEM is the receptor for multiple ligands including the TNF fam-ily proteins LIGHT (also known as HEVMLTNFSF14) and lymphotoxin-α and the immunoglobulin superfamily (IgSF) proteins B and T lymphocyte attenuator (BTLA) and CD160 LIGHT and lymphotoxin-α deliver a co-stimulatory signal to HVEM+ cells By contrast BTLA and CD160 bind to HVEM and deliver an inhibitory signal shutting down T cell activation BTLA expres-sion mirrors that of HVEM with the kinetics of expres-sion being reversed thus although HVEM is highly expressed on naive T cells and is reduced upon activa-tion BTLA is expressed upon activation presumably to attenuate T cell activity CD160 functions similarly to control T cell responses through HVEM but can itself transduce inhibitory signals to T cells presumably act-ing in trans96 HVEM therefore functions as a bimodal switch with the cellular response of activation or inhibi-tion depending on which ligand is engaged

Numerous studies have shown that BTLA blunts antitumour T cell activation by signalling through HVEM9798 The importance of BTLA signalling is

Box 4 | Biomarkers for checkpoint inhibitor responses

Although some pharmacodynamic markers are suggestive predictive biomarkers have been unable to define which subset of patients will benefit from cytotoxic T lymphocyte antigen 4 (CTLA4) blockade33125 Considerable effort has been dedicated to the analysis of programmed cell death 1 ligand 1 (PDL1) expression as a predictive biomarker for programmed cell death protein 1 (PD1)ndashPDL1 inhibitors (reviewed in REF 225) Some tumours may overexpress PDL1 secondary to genomic amplification as seen in Hodgkin lymphoma226 This may account for the exceptionally high response rates recently reported in patients with treatment-refractory Hodgkin lymphoma treated with nivolumab227 However PDL1 expression in most tumours is not a function of genomic amplification PDL1 expression on tumour cells is the most robust marker associated with response to the PD1-specific mAb nivolumab in melanoma whereas multiple inflammatory markers (PD1+ tumour-infiltrating lymphocytes tumour cell expression of PDL2) also correlate with response228 In a Phase I trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) high PDL1 expression on tumours was associated with greater response11 In a Phase III trial of patients with nonsquamous NSCLC who were randomized to nivolumab or docetaxel patients whose tumours expressed high levels of PDL1 had greater responses than those with low PDL1 expression229 whereas PDL1 expression on squamous NSCLC did not appear to have a significant role as a biomarker230 Recent reports of predictive correlates of response to the PDL1-specific mAb atezolizumab support that high levels of PDL1 expression within the tumour especially by tumour-infiltrating immune cells is associated with response to treatment710 In some clinical trials such as with atezolizumab and pembrolizumab PDL1 expression is used as a biomarker to enrich the patient population for potential responders (ClinicalTrialsgov identifiers NCT01375842 NCT01848834) The role of PDL1 expression as a prognostic or predictive biomarker in different tumour types requires further evaluation in the context of a randomized clinical trial

Although PDL1 status may be useful for predicting the subset of patients who are more likely to respond to single-agent PD1 pathway blockade objective response rates of 5ndash20 in PDL1-negative tumours have been reported across tumour types The PDL1 status of a tumour may be an imperfect biomarker that is not specific to PD1 pathway blocking agents but indicative of a smouldering immune response that can be made effective by other immune-stimulating agents such as interleukin-2 (IL-2) an engineered IL-2 variant231 or other agonist therapies Be it on tumour cells or the immune cells infiltrating into the tumour PDL1 expression may be one component of a more powerful predictive model to differentiate which patients would benefit from immune checkpoint monotherapy combination therapy or standard therapies A two-variable model incorporating both PDL1 expression and lymphocyte infiltration has been proposed for discriminating tumours more likely to respond to immune checkpoint blockade232 Given the significant response rates seen in patients with PDL1-negative tumours many researchers and clinicians feel that PDL1 expression is not an appropriate biomarker to exclude patients from receiving the immune checkpoint inhibitors Notably combining nivolumab and ipilimumab appeared to improve the response rates of PDL1-negative melanoma greater than PDL1-positive melanoma16 which suggests that PDL1 status may best be used as a means of directing patients less likely to respond to monotherapy to immune checkpoint combinations rather than exclusion from immune checkpoint therapy Clearly combination therapies are the next logical step to improve response rates increase duration of responses and potentially increase cure rates The development of prognostic and predictive biomarkers of response need dedicated clinical trials and will guide patient selection for either monotherapy or specific therapeutic combinations Given the multiple immunosuppressive factors on the tumour and immune cells PDL1 expression is likely to be only one of many variables in the predictive matrix for immune-based therapies

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NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

580 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 12: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

NY-ESO-1A tumour antigen that is expressed on various tumours including some melanoma It may be used to track the immune response to the tumour by identifying NY-ESO-1-specific T cells using NY-ESO-1 peptidendashMHC class I tetramers

illustrated by the phenotype of the BTLA-deficient mouse which develops severe autoimmunity99 BTLA is overexpressed on diverse tumour types suggesting that this pathway has been co-opted for use in immune evasion BTLA antagonism can be achieved by targeting the BTLACD160-binding domain of HVEM which is in the first cysteine-rich domain (CRD1) Importantly this domain is not required for binding the stimulatory ligands LIGHT and lymphotoxin-α96 An alternative strategy is to target BTLA and CD160 with antagonist antibodies to block binding Given these observations therapeutics targeting the HVEM axis are likely to see clinical development in the near future Patent filings suggest that active programmes are under development by Bristol-Myers Squibb and INSERM (the French Institute of Health and Medical Research)

In summary the TNFR family provides T cells natural killer cells and myeloid cells with diverse recep-tors that have both overlapping and distinct activities Improved understanding of their gene expression in

individual cancers will help direct which tumours would benefit from combination agonist therapy or combina-tion of an agonist with one or more immune checkpoint inhibitors One promising path through this complex-ity is offered by the analysis of tumour-infiltrating lym-phocytes It is apparent that the mechanism of tumour immune evasion varies across tumour types among patients with the same tumour and within a given patientrsquos tumour (temporally and spatially) Profiling tumour-infiltrating lymphocytes peripheral blood mon-onuclear cells and sentinel lymph nodes for the expres-sion patterns of immune checkpoint and TNFR proteins will help to guide combination therapy

Novel IgSF proteinsSeveral cell surface proteins within the IgSF family have been identified that regulate T cell responses Some are expressed on exhausted T cells (for example PD1) and some are expressed by the tumour itself (for example PDL1) Notably none of the more recently discovered immunosuppressive IgSF immune checkpoint proteins is closely related to the canonical pathways CTLA4ndashCD80 (also known as B7-1)ndashCD86 (also known as B7-2) or PD1ndashPDL1ndashPDL2 (also known as B7-DC)

LAG3 LAG3 which was cloned in the early 1990s as a CD4 homologue is a good example LAG3 was identi-fied as a selective marker of TReg cells100 Critically LAG3 was shown to confer TReg cell function when transfected into naive CD4+ T cells100 Binding of both CD4 and LAG3 to the MHC class II protein is required for pro-ductive T cell interaction with APCs This result suggests that LAG3 functions as a negative regulator of T cell responses to MHC class II-restricted antigen presenta-tion LAG3 is co-expressed on exhausted CD8+ T cells and antibody-mediated antagonism of LAG3 and PD1 synergistically reactivated exhausted CD8+ T cells in a chronic viral infection model5 Mice deficient in both LAG3 and PD1 are able to reject even poorly immu-nogenic tumours illustrating the non-overlapping role of these proteins in regulating immune responses101 Encouragingly combination blockade is effective and tolerable in preclinical models however caution is war-ranted as mice deficient in both LAG3 and PD1 develop aggressive autoimmunity that is more severe than either single-gene deficiency101

Studies utilizing tumour-infiltrating lympho-cytes from patients with ovarian cancer found that LAG3+PD1+ CD8+ T cells that recognize the antigen NY-ESO-1 were impaired in their ability to respond to antigen stimulation102 Although LAG3 inhibition alone was not sufficient to restore antigen-specific T cell responsiveness the combined blockade of LAG3 and PD1 was more effective than PD1 blockade alone These data suggest that in tumours in which LAG3 and PD1 are co-expressed on tumour-infiltrating lymphocytes dual therapy may be an effective means of increasing response rates andor the effectiveness of therapy

Multiple companies have developed LAG3-specific antagonist antibodies including Bristol-Myers Squibb Novartis and TesaroAnaptysBio A LAG3ndashFc fusion

Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cells Regulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T lymphocyte antigen 4 (CTLA4)-specific mAb ipilimumab may function in part through the depletion of regulatory T

cells which

constitutively express cell surface CTLA4 CD25 also known as interleukin-2 receptor subunit-α GITR gluco corticoid-induced TNFR-related protein ICOS inducible T cell co-stimulator LAG3 lymphocyte activation gene 3 protein PD1 programmed cell death protein 1 TNFRSF25 tumour necrosis factor receptor superfamily member 25 VEGFR vascular endothelial growth factor receptor

Nature Reviews | Drug Discovery

Regulatory T cell

LAG3

TNFRSF25

GITR

4-1BB

OX40

CD27

ICOS

CD25

CTLA4

PD1

VEGFR

Neuropilin

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protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 577

copy 2015 Macmillan Publishers Limited All rights reserved

based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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copy 2015 Macmillan Publishers Limited All rights reserved

antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 13: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

protein IMP321 (developed by Immutep) is in clinical development and has shown biological activity and clinical responses in renal cell carcinoma metastatic breast cancer and advanced pancreatic cancer103ndash105 The LAG3-specific antibody BMS986016 (developed by Bristol-Myers Squibb) Phase I trial in advanced solid tumours has a nivolumab combination arm (NCT01968109) TesaroAnaptysBio have bi-specific antibodies targeting both LAG3 and PD1 in preclini-cal development However the mechanism of action of LAG3-targeted agents on CD8+ T cells remains unclear as LAG3 binds to MHC class II whereas CD8+ T cell responses are MHC class I-restricted

TIM3 Another T cell-expressed IgSF protein of sig-nificant interest is TIM3 a receptor within the subfamily of TIM (T cell-immunoglobulin-mucin domain) pro-teins106 The potency of the TIM3 pathway was apparent from research conducted using TIM3 antagonists and a Tim3-deficient mouse model including transplanta-tion autoimmunity and antigen challenge models107 For example in chronic viral infection models the expres-sion of TIM3 and PD1 identified highly dysfunctional CD8+ T cells that were unable to respond to antigen stimulation108

The identification of TIM3+PD1+ CD8+ tumour-infiltrating lymphocytes as exhausted T cells indi-cated the importance of TIM3 in the cancer setting Subsequent work in preclinical animal models using syngeneic tumour challenge models demonstrated the utility of TIM3 antagonism In these models all TIM3+ infiltrating T cells also expressed PD1 (REF 109) and the antitumour efficacy of TIM3 antagonism was enhanced in combination with PD1 blockade TIM3 antagonism was also enhanced when combined with an agonist 4-1BB-specific antibody76

Multiple TIM3 ligands have been described including phosphatidylserine galectin 9 (GAL9) high mobility group protein B1 (HMGB1) and carcinoem-bryonic antigen-related cell adhesion molecule 1 (CEACAM1)107110111 Because the blocking character-istics of therapeutic TIM3-specific mAbs are incom-pletely described their mechanism of action is poorly understood Just as blocking the PD1ndashPDL1 pathway may affect cell types other than T lymphocytes blocking TIM3 may have the same effect because it is expressed on multiple haematological cells Human TIM3 is expressed by various T cell populations natural killer cells natural killer T cells and APCs such as dendritic cells and macrophages Because TIM3 is capable of sup-porting phosphatidylserine-mediated phagocytic activ-ity as shown in TIM3 gain-of-function experiments it may be functioning by a markedly different mechanism from previously mentioned IgSFs It is a reasonable hypothesis that blocking TIM3 in the tumour response setting is efficacious because the ability of the immune system to recognize dead and dying tumour cells is altered112

A role for TIM3 interaction with GAL9 in the control of T cell responses to tumour cells has also been pro-posed113 We propose that GAL9 which binds to specific sugar residues on multiple glycoproteins such as 4-1BB

and TIM3 may act as a general enhancer of glyco protein receptor signalling by increasing crosslinking after the canonical ligand is engaged114 TIM3 has recently been recognized as a receptor on natural killer cells115 although the importance of this finding for immune checkpoint therapy has yet to be demonstrated This raises the important question of when it is clinically rel-evant to administer anti-TIM3 therapy The preclinical data suggest that co-expression of TIM3 with PD1 andor LAG3 on tumour-infiltrating lymphocytes may be the relevant indication that dual therapies are warranted109 Although there are no TIM3 antagonists in clinical trials several are in preclinical development

Siglecs Sialic acid-binding immunoglobulin-type lectins (siglecs) are a class of IgSF proteins that bind sialic acid (FIG 4) They are found primarily on haematopoietic cells and are widely expressed on lymphocytes and APCs116 Sialic acid-rich glycoproteins are often highly expressed on metastatic cancer cells Siglec 7 and siglec 9 are over-expressed on various solid tumours and block natural killer cell-mediated antitumour cytotoxicity suggesting that antagonists of these siglecs might have therapeutic benefits117 A genetic polymorphism that reduces siglec 9 binding to tumours was associated with favourable out-comes in patients with early-stage NSCLC118 Gene defi-ciency of siglec E (the murine homologue of siglec 9) led to an increased antitumour response in vivo118 Siglec 15 engagement on MDSCs by tumour-associated sialyl-TN antigen induced the secretion of TGFβ indicating a role for siglec 15 in the establishment or maintenance of the immune suppressive tumour microenvironment119 As there are 14 human siglecs and diverse siglec ligands additional data on the role of this family in modulating antitumour immunity is anticipated

B7 and CD28-related proteinsRecent studies have highlighted the role of IgSF pro-teins that are related to the CD28 and CTLA4 ligands CD80 and CD86 (B7-2) including B7-H3 (also known as CD276) B7-H4 (also known as VTCN1) V-domain immunoglobulin suppressor of T cell acti-vation (VISTA) HERV-H LTR-associating protein 2 (HHLA2) and other proteins expressed on immune cells and often on tumour cells120ndash122 (FIGS 12) Butyrophilins and butyrophilin-like molecules comprise a diverse family of IgSF proteins that are structurally related to B7 proteins Although interesting preclinical data have accumulated for each of these targets there are limited clinical trial data

ICOS Although the majority of the IgSF family mem-bers described below suppress T cell and in some cases natural killer cell activity the CD28 family IgSF protein inducible T cell co-stimulator (ICOS) is expressed on activated T cells and mediates T cell activation ICOS is activated by ICOS ligand which is expressed on B cells and APCs123 However little work has been conducted to develop agonist agents to target ICOS perhaps because of the disastrous consequences of targeting the related CD28 pathway with an activating antibody as

PhosphatidylserineA phospholipid in the cell membrane normally found on the inner surface unless the cell is undergoing cell death in which it can be found on the cell surface and serves as an lsquoeat mersquo signal for phagocytes A ligand for the T cell immunoglobulin mucin 3 immune checkpoint

Galectin 9(GAL9) A member of a family of proteins that can bind β-galactoside sugars Some galectins are overexpressed by tumour cells and some can crosslink extracellular proteins (for example 4-1BB T cell immunoglobulin mucin 3) and potentiate their immunomodulatory function

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described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 14: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

described earlier19 ICOS has been receiving renewed interest as a marker of immunocompetent T cells within tumours124 In patients with melanoma receiving ipili-mumab an increase in ICOS+ CD4+ T lymphocytes is a pharmacodynamic marker and may predict response125 Although ICOS expression is also associated with TReg cell activity ICOS activation had synergistic effects with CTLA4 blockade in preclinical models of melanoma and pancreatic cancer126 Agonist ICOS-specific anti-bodies and ICOS ligand constructs (for example ICOS ligand-Fc) are visible in the patent databases notably from INSERM the Memorial SloanndashKettering Cancer Center and Bristol-Myers Squibb Jounce Therapeutics has an agonistic ICOS-specific mAb that is at the lsquoIND-enablingrsquo phase (as recently presented at PEGS Boston) but no therapeutics have reached the clinic yet

B7-H3 and B7-H4 B7-H3 and B7-H4 are interesting examples of the B7-related proteins they also show modest homology with PDL1 and PDL2 (REF 127) Most studies support an immunoinhibitory function for these proteins and their expression on multiple solid tumour types makes them attractive anticancer candidates (TABLE 1) However the biology of B7-H3 and B7-H4 is incompletely understood and their ligands are uniden-tified therefore targeting these proteins is challenging Macrogenics has developed a depleting antibody that targets B7-H3 but as this is not an immune checkpoint approach we will not cover it here Further understand-ing of the biology of B7-H3 and B7-H4 will certainly further accelerate drug development

VISTA From the perspective of immune checkpoint combinations VISTA seems particularly attractive VISTA is highly expressed on myeloid cells and TReg cells As seen with PD1 blockade VISTA-specific anti-body blockade increased the infiltration proliferation and effector function of tumour-infiltrating lymphocytes in a melanoma model while blocking TReg cell function VISTA blockade also decreased the number of MDSCs and increased the presence of activated dendritic cells within the tumour microenvironment128 With these results we are beginning to see a potentially impor-tant impact on non-T cell populations as effects on the MDSC population might synergize with therapeutics designed to unleash effector T cells such as antibodies against CTLA4 and PD1 or agonist antibodies to TNFRs

HHLA2 Another B7-related protein of potential interest is HHLA2 which inhibits the proliferation of both CD4+ and CD8+ T cells and reduces the production of IFNγ TNFα IL-5 IL-10 IL-13 IL-17A and IL-22 by T cells122 HHLA2 is widely expressed in solid tumours including breast lung melanoma pancreatic ovarian and others making it an attractive therapeutic target in a manner similar to PDL1 (REF 129) Furthermore in breast cancer ~30 of patients tested showed HHLA2 gene amplifica-tion129 Transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2) which is expressed on T cells has been identified as a receptor for HHLA2 however crosslinking of TMIGD2 stimulates T cell

activation130 These conflicting reports of inhibitory or stimulatory activity might be explained by multiple receptors or different experimental systems Additional work is anticipated but will not come from murine studies as HHLA2 has no orthologue in mice or rats

BTNL2 Butyrophilin-like protein 2 (BTNL2) is a butyrophilin that is highly expressed in lymphoid and mucosal tissues A BTNL2-Fc fusion protein was shown to downregulate the response of T cells to antigen stimu-lation131 A polymorphic variant of BTNL2 has a muta-tion in a splice site that leads to a truncated protein that is associated with several inflammatory diseases including sarcoidosis and myositis SNP analyses have linked the gene to many autoimmune and chronic inflammatory conditions132133 Additional genetic studies have associ-ated BTNL2 with diverse cancer indications including prostate cancer and marginal zone lymphoma134135 As is typical of the butyrophilins no protein ligand has been identified although a BTNL2-Fc fusion protein was shown to bind to activated lymphocytes131 As the genetic data are compelling we suspect it is only a ques-tion of time before we see reagents targeting this path-way to enhance immune responses although the region on BTNL2 to target is unknown

Other butyrophilin family members with low but rec-ognizable homology to the B7 and PD1 ligands include approximately 30 more identified genes Butyrophilin activity in immune responses has been recently reviewed and provides additional targets of interest133136

TIGIT and PVR family membersAnother protein with diverse functional activity is TIGIT (T cell immunoglobulin and ITIM domain) which similar to PD1 is expressed on exhausted CD8+ T cells but also on natural killer cells (FIG 4) TIGIT is a member of the poliovirus receptor (PVR) family within the IgSF that also includes CD226 (also known as DNAM1) CD96 CD112 (also known as PVRL2nectin 2) CD155 (also known as PVR) and related proteins such as nec-tins137 These proteins interact with each other to influ-ence natural killer cell and T cell immune activities and TIGIT blockade worsens autoimmune diseases in mouse models138

TIGIT binds CD155 CD112 and the related nectin family member CD113 (also known as PVRL3nectin 3) (FIG 4) Tigit knockout mice show delayed tumour growth in a syngeneic melanoma model an effect that can be traced in part to expression of TIGIT on TReg cells139 However much of the activity ascribed to TIGIT involves the interaction of TIGIT on T cells and perhaps natural killer cells with CD155 expressed on immature or resting dendritic cells which blocks maturation sig-nals normally delivered by CD226 TIGIT binds CD155 with a higher affinity than CD226 which means that immunoinhibition will override activation as is typical for negative regulatory signals

A recent study described TIGIT expression on tumour antigen-specific CD8+ tumour-infiltrating lymphocytes isolated from patients with melanoma140 TIGIT+ cells often co-expressed PD1 and downregulated

Poliovirus receptor(PVR) A protein that is expressed by dendritic cells but may be overexpressed by some tumours and can bind T cell immunoglobulin and ITIM domain (TIGIT) a related protein expressed on T cells and natural killer cells

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the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 577

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 15: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

the expression of CD226 perhaps supporting a prefer-ential interaction of TIGIT with CD155 and CD112 (FIG 4) Notably these TIGIT ligands are upregulated by tumour cells and associated myeloid and dendritic cells in the tumour When CD155 and CD112 were present co-blockade of TIGIT and PD1 additively increased T cell proliferation cytokine production and granzyme B production suggesting that combination therapy might be useful in tumours showing high expression of TIGIT CD155 and CD112

Genentech is developing TIGIT-specific therapeu-tics and recently showed that co-blockade of TIGIT and PDL1 resulted in tumour rejection by restoring the func-tion of exhausted tumour-infiltrating CD8+ T cells141 The mechanism of action of this effect appears to be antagonism of TIGIT-mediated blockade of CD226 function Although intriguing these in vivo studies used very high amounts of ADCC-competent TIGIT-specific antibody which can complicate interpretation (BOX 3)

CD226 was recently implicated in immune surveil-lance in a transgenic multiple myeloma mouse model142 When the multiple myeloma transgenic mice were crossed with Cd226-deficient mice spontaneous develop-ment of multiple myeloma was reduced Immune surveil-lance and tumour control in this model required CD226 expression on natural killer and CD8+ T cells that secreted perforin and IFNγ Additional experiments showed that CD226 expression was required for optimal anti-myeloma efficacy of cyclophosphamide and bortezomib which

are the standard of care for patients with multiple mye-loma In mice a TIGIT-specific mAb synergized with a TIM3-specific mAb to reduce tumour growth139

These studies support a critical role for the TIGITndashCD226 axis in regulating antitumour immunity The early data accumulating for TIGIT and interacting pro-teins suggest that targeting TIGIT may be particularly beneficial in tumour settings in which both T cells and natural killer cells have therapeutic potential These early data also suggest that development of therapeu-tics should be conducted cautiously given that TIGIT engages in multiple protein interactions (FIG 4) Indeed the finding that TIGIT loss-of-function mutations in humans are associated with dysregulated triglyceride levels suggests that there are additional functions of this protein yet to be elucidated143

A critical issue to address as we examine the immu-nological universe for additional therapeutic targets is the development of guidelines for their rational combi-nation and incorporation into the standard of care par-ticularly in the treatment of cancers beyond melanoma To the extent that combination therapy may engage dif-ferent arms of the immune response they should show additive or synergistic effect Understanding patterns of expression of ligands and receptors within the tumour on tumour-associated cells in tumour-draining lymph nodes and in the circulation will be critical to developing rational combinations between IgSF-directed therapeu-tics and with other immunomodulatory agents

Figure 4 | Activating and inhibitory receptors on natural killer cells Natural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For example engagement of TIGIT (T cell immunoglobulin and ITIM domain) or KIR (killer inhibitory immuno-globulin-like receptor) on NK cells can inhibit (red) their function whereas engagement of NKG2D can activate (green) NK cell function GM-CSF granulocytendashmacrophage colony-stimulating factor IFNγ interferon-γ ILT immunoglobulin-like transcript MICA MHC class I polypeptide-related sequence A RAET1ULBP retinoic acid early transcripts 1UL16-binding protein TNFα tumour necrosis factor-α

Nature Reviews | Drug Discovery

Antigen presenting cellTumour cell NK cell

CD96

TIGIT

CD226

ILT family

KIR

CD244 CD48

CD112

CD155

Siglecs family

NKG2D

bull IFNγbull TNFαbull GM-CSF

MHC class I

Sialylated glycan

MICA family

RAET1ULBP family

Activation

Inhibition

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Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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copy 2015 Macmillan Publishers Limited All rights reserved

antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 16: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

Immunomodulatory targets on natural killer cellsThe natural killer cell is a critical mediator of innate immune responses via direct cell killing and rapid cytokine production (FIG 4) There is abundant evidence showing that the innate immune system contributes to the antitumour immune response Tumour cells can downregulate MHC class I to avoid recognition by CD8+ T cells however this can make tumour cells more sus-ceptible to lysis by natural killer cells IFNα therapy can effectively control some tumour types by direct activation of innate immunity albeit in a minority of patients

Evidence that natural killer cell and T cell antitumour activities are complementary in the immunotherapy set-ting comes from recent studies exploring natural killer cell-specific cell surface proteins such as the killer inhibi-tory receptors Although clinical trials have explored the role of natural killer cell-based vaccine therapies argua-bly the potential of the natural killer cell is best illustrated by therapeutic mAbs that mediate their effect by ADCC activation of natural killer cells through its Fc receptor For example in women with metastatic breast cancer response to the HER2Neu (also known as ERBB2)-targeted antibody trastuzumab (Herceptin Roche) cor-relates with natural killer cell activity144 Natural killer cell stimulating therapy may therefore improve responses to ADCC-dependent therapies Moreover combining T cell and natural killer cell-directed therapies may recruit the strength of each therapy mdash maximal cytotoxicity and the establishment of immunological memory

Killer inhibitory receptors Killer inhibitory receptors include the KIRs and C-type lectin transmembrane receptors KIRs interact with cell surface human leukocyte antigen (HLA) proteins to inhibit natural killer cell activa-tion Lirilumab is an antagonist antibody that specifically recognizes KIR2DL1 KIR2DL2 and KIR2DL3 and pre-vents inhibitory signalling triggered by binding to HLA-C thereby increasing natural killer cell-mediated killing of HLA-C-expressing tumour cells Lirilumab had only modest toxicity and showed signs of clinical activity in Phase I trials145 Although lirilumab monotherapy did not have any dose-limiting toxicities a recent update of the Phase I trial reported toxicity across a wide dose range (up to 10 mg per kg) with more than 50 of patients report-ing grade 3ndash4 toxicity even at the 015 mg per kg dose71 This clearly illustrates the importance of vigilant dose-finding trials as combination trials with lirilumab are currently ongoing Phase I trials of combination treatment with nivolumab and lirilumab for the treatment of solid tumours have begun (NCT01714739 NCT01592370) A combination trial of lirilumab or urelumab with the SLAM7-specific depleting antibody elotuzumab is in progress for multiple myeloma (NCT02252263) A recent update to clinical trial filings indicates that lirilumab com-binations with nivolumab have been added as cohorts in clinical trials of ipilimumab with nivolumab for haema-tological malignancies (NCT01592370)

C-type lectins C-type lectins are a large family of pro-teins that bind to select carbohydrates (for example man-nose fucose and glycan) C-type lectins are found on

macrophages dendritic cells natural killer cells and other cell types that respond to pathogens and can transduce stimulatory or inhibitory signals Among the C-type lec-tins the NKG2D and NKG2A proteins potently modulate natural killer cell and CD8+ T cell function NKG2D acts as a co-stimulatory protein on T cells augmenting CD28 and T cell receptor signalling and directly activating natu-ral killer cells via engagement with HLA-related ligands of the UL16-binding protein (ULBP) family (ULBP1-6) and the MHC-like highly polymorphic proteins MHC class I polypeptide-related sequence A (MICA) and MICB146147 MIC proteins are normally expressed on the gastrointes-tinal epithelium endothelial cells and fibroblasts Stressed or highly proliferative cells can also upregulate MICA and MICB and tumour cells of diverse origin including hae-matological cancers express these proteins148 Chronic MICA overexpression may contribute to immune eva-sion by disrupting NKG2D activity on natural killer cells and perhaps on T cells147 This effect is exacerbated by the aggressive shedding of MICA from tumour cell surfaces by the ADAM family of proteases which are themselves overexpressed by tumour cells IPH43 (developed by Innate Pharma) is a MICA-specific antibody that binds to nearly all polymorphic classes of MICA This antibody neutralizes soluble MICA blocks the interaction of MICA with NKG2D and may also directly induce ADCC and complement-dependent cytotoxicity-mediated killing of MICA-expressing tumour cells149 Multiple clinical trials of IPH43 are underway

NKG2A is an inhibitory receptor on natural killer cells and T cells that blocks the interaction of HLA-E ligands with the activating receptor NKG2C150 NKG2A is co-opted by tumour cells as an immune checkpoint receptor that inhibits antitumour activity functions of cytotoxic natural killer and T lymphocytes via overex-pression of HLA-E This mechanism is observed across diverse tumour types including both solid tumours and haematological malignancies151 The NKG2A-specific antibody IPH2201 (developed by Innate Pharma) blocks the inhibitory function of NKG2A thereby facilitating natural killer cells and T cells to mount an antitumour immune response Phase III trials with IPH2201 are ongoing in cancer indications (NCT02331875)

ILTLIR The immunoglobulin-like transcriptleuko-cyte immunoglobulin-like (ILTLIR) protein family is closely related to the KIRs ILTs are also inhibitory recep-tors that interact with the MHC to regulate immune responses Indeed the ILT ligand HLA-G is highly upregulated on fetal cells of the placenta and prevents a natural killer cell response to MHC class I-negative fetal cells152 During inflammation HLA-G+ dendritic cells induced by immunosuppressive molecules such as IL-10 and IDO actively tolerize diverse immune cells by bind-ing to members of the ILT family153 HLA-G expression has been detected in a wide variety of human cancers including both solid tumours and haematological malig-nancies Importantly HLA-G can be shed from the cell surface and soluble HLA-G functionally engages ILTs High levels of HLA-G in plasma are a marker of poor prognosis in many tumour types154

Human leukocyte antigen(HLA) The genetic designation for the human MHC

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CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 577

copy 2015 Macmillan Publishers Limited All rights reserved

based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

580 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 17: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

CD244 and CD48 Triggering of CD244 (also known as natural killer cell receptor 2B4) by CD48 induces natu-ral killer cell cytotoxic activity resulting in the killing of target cells155 Engagement of CD244 on human natural killer cells by a CD244-specific agonist mAb induced IFNγ secretion156 and some models suggest that the natu-ral killer cell response to tumour cells is increased if those cells express CD48 Although CD48 is clearly an activating ligand for CD244 on human natural killer cells the means by which this pathway is regulated remain unclear as CD48 is expressed on nearly all haematopoietic cell types It has also been suggested that CD244ndashCD48 signalling is bi-directional that is CD48 signals when bound to CD244 (REF 155) Because CD48 is a glycophosphatidylinositol-linked protein that lacks intracellular signalling domains such a mechanism requires further study Thus although this pathway remains of significant interest because it appears to directly engage the natural killer cell cytotoxic-ity machinery further work is required to understand how best to modulate this system for antitumour therapy157

Critical issues to address include how and when to use the natural killer cell-directed therapeutics Clearly those tumour types that actively deploy natural killer cell evasion strategies would be most likely to benefit In addition downregulation of MHC class I is an obvious mechanism by which a tumour could become resistant to the CD8+ T cells stimulated by therapies targeted at PD1 or CTLA4 This evasion strategy should make the tumour more susceptible to natural killer cell lysis and suggests that natural killer cell therapies might be started after checkpoint blockade is established In this regard we can hope for the development of biomarker strategies that identify susceptibility to killing by natural killer cells alongside the development of novel therapeutics

Targeting the tumour microenvironmentOur focus thus far has been largely on cellndashcell inter-actions and more specifically lymphocytendashAPC and lym-phocytendashtumour interactions There are also myriad and complex mechanisms for cellular regulation mediated by soluble andor secreted molecules (FIG 5)

IDO The cytosolic enzyme IDO1 is the rate-limiting enzyme for the catabolism of the essential amino acid tryptophan In the tumour microenvironment IDO1 is produced by tumour cells and by tumour-associ-ated MDSCs and tumour-associated macrophages in response to inflammatory signals such as IFNγ High levels of IDO1 reduce tryptophan levels and create bio-active tryptophan metabolites both of which suppress T cell activity158 Preclinical data supports the hypothesis that tumours use IDO1 to suppress immune responses as IDO1 inhibition led to tumour control in a variety of syngeneic mouse models158

The IDO1 inhibitor INCB024360 (developed by Incyte) is currently in Phase I and II trials for metastatic melanoma in combination with ipilimumab and as mon-otherapy for ovarian cancer In the ipilimumab combi-nation study many patients demonstrated an objective response and maintained stable disease159 INCB24360 is also being studied in a Phase III trial in advanced or metastatic cancers including melanoma and NSCLC in combination with pembrolizumab Other clinical tri-als with INCB24360 include combination studies with nivolumab and the PDL1-specific antibodies atezoli-zumab and MEDI4736 There are also clinical studies with indoximod (developed by NewLink Genetics) a tryptophan analogue IDO1 inhibitor The mechanism of action of indoximod is not well understood as it does not appear to influence free tryptophan levels The company has presented Phase I trial data showing good tolerability and some early signals of clinical activ-ity (NCT00739609)160 This programme was recently partnered with Roche161 Flexus Biosciences developed a comprehensive preclinical program targeting IDO and tryptophan regulatory pathways the company was recently acquired by Bristol-Myers Squibb162

TGFβ TGFβ is a pleotropic cytokine with multiple tumour-supporting effects including angiogenesis and immunosuppression Increased TGFβ expression is strongly associated with progression and poor clini-cal outcome in diverse tumour indications TGFβ requires cleavage for activation a carefully orchestrated process involving extracellular matrix adhesion and G protein-coupled receptor-mediated integrin activa-tion163 Importantly TGFβ production is induced in cells associated with the tumour stroma reflecting the close association of TGFβ cleavage with integrin acti-vation These properties make TGFβ a compelling therapeutic target but one requiring careful design The most advanced TGFβ-targeting drug in clinical devel-opment is LY2157299 (galunisertib developed by Eli Lilly and Company) a small-molecule kinase inhibitor designed to selectively block TGFβ receptor signalling Galunisertib has a well-defined therapeutic window

Figure 5 | Immunosuppressive factors in the tumour microenvironment In addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating effector cells within the tumour Immunosuppressive factors can be secreted by tumour cells stromal cells (such as fibroblasts) or infiltrating myeloid cells IDO indoleamine 23-dioxygenase IL-10 interleukin-10 MDSC myeloid-derived suppressor cell TGFβ transforming growth factor-β VEGF vascular endothelial growth factor

Nature Reviews | Drug Discovery

Myeloid-derivedsuppressor cell

Tumourcell

Fibroblast

bull VEGFbull Adenosinebull IDObull IL-10bull TGFβbull Galectins

Tumourndashassociatedmacrophage

IntegrinA protein that is specialized for cellndashcell or cellndashextracellular matrix interactions

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 577

copy 2015 Macmillan Publishers Limited All rights reserved

based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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copy 2015 Macmillan Publishers Limited All rights reserved

antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 18: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

based on dampening but not shutting down receptor signalling164 and has shown clinical activity in a variety of solid tumour models This and other TGFβ inhibi-tors have been tested in combination therapy in pre-clinical models showing synergy with other therapeutic modalities such as chemotherapy165 and growth factor inhibition166 In a transgenic mouse melanoma model galunisertib showed synergistic activity with anti-CTLA4 therapy167 A collaboration to study galunisertib in com-bination with nivolumab in advanced solid tumours has recently been announced by Bristol-Myers Squibb and Eli Lilly and Company Newer approaches to targeting TGFβ include blocking local activation of the cytokine as it is now appreciated that the mechanisms of TGFβ activation are diverse and highly selective for different microenvironments163

Adenosine Extracellular adenosine can be detected by receptors that regulate immune functions One of the signature features of the solid tumour microenviron-ment is hypoxia a state of cellular oxygen deprivation that induces VEGF production angiogenesis and diverse epigenetic changes in tumour cells including the regu-lation of survival signals and changes in metabolism168 Hypoxia also causes an accumulation of extracellular adenosine that signals through the adenosine receptor A2A (ADORA2A) ADORA2A is expressed on CD8+ tumour-infiltrating lymphocytes natural killer cells and MDSCs and is an important component of immune reg-ulation helping to stop immune responses in the con-text of inflammation120 Multiple lines of evidence suggest that targeting the adenosinendashADORA2A pathway will be efficacious in the setting of tumour immunotherapy ADORA2A deficiency or pharmacological blockade has been shown to decrease the immunosuppression of CD8+ tumour-infiltrating lymphocytes in multiple preclinical models169170 including an increase in IFNγ secretion by these cells Although targeting ADORA2A directly is one approach to modulate this pathway the complexity of ADORA2A biology which includes effects on the CNS and peripheral physiology and pathology has encour-aged the development of alternative strategies171

Extracellular adenosine can be produced by the ectonucleotidases CD39 (also known as ectonucleo-side triphosphate diphosphohydrolase 1) which con-verts ATP to ADP and ADP to AMP and CD73 (also known as 5ʹ-nucleotidase) which converts AMP to adenosine172 Both ectonucleotidases can be upregu-lated on tumour cells and also on tumour-associated TReg cells173 A proof-of-concept study using a CD73 inhibitor demonstrated additive antitumour activity with CTLA4-specific antibody treatment in a melanoma model174 A CD73-specific antibody or hyperoxia had additive activity when combined with CTLA4-specific or PD1-specific antibodies in multiple tumour mod-els175176 This study demonstrated particularly strong additive activity in the setting of anti-PD1 therapy pos-sibly because adenosine upregulated PD1 expression on the target tumour cells These two studies focused on the effect of adenosine production and signalling through ADORA2A on CD8+ T cells Emerging data suggest that

ADORA2A signalling also triggers MDSCs to promote immunosuppression and that natural killer cells are negatively regulated via this pathway177ndash179 Clinical trials are several years away

The hypoxic environment of tumours induces myriad other pathways and modulators180 Of recent and intensive interest are the chemokine receptors nota-bly CXCR4 cytokines including IL-10 and IL-27 and growth factors such as VEGF as discussed earlier With or without hypoxia tumour-associated cell popula-tions are critical to tumour proliferation resistance to therapy and even metastasis Successful targeting of MDSCs tumour-associated macrophages and familial adenomatous polyposis (FAP)+ fibroblasts will require a better understanding of the dependencies of those par-ticular cell types For example inhibition of the CXCR4 ligand CXCL12 synergized with anti-PDL1 therapy in pancreatic cancer due to antagonism of FAP+ fibroblast immunosuppressive activities181

Targeting cell discrimination mechanismsThe immune system uses diverse mechanisms to identify problem cells be they dying cells that need to be cleared by macrophages infected cells that need to be eliminated by immune responses or tumour cells that are prolifer-ating unnaturally Tumour cells have co-opted mecha-nisms for subverting cell discrimination mechanisms to blunt the ability of the innate and adaptive immune responses to recognize them

Phosphatidylserine Phosphatidylserine is a membrane marker exposed during the process of programmed cell death or apoptosis and serves as an lsquoeat mersquo signal for phagocytes Phosphatidylserine is also expressed on rapidly dividing cells notably proliferating lymphocytes tumour cells and the tumour vasculature Profligate tumour cell proliferation leads to the death of large num-bers of cells Because apoptosis is a controlled mecha-nism of cell death it does not normally trigger immune activation and therefore phosphatidylserine expression on tumour cells does not induce an antitumour immune response Indeed tumour cells may further evade infil-trating immune cells via binding of phosphatidylserine to TIM3 expressed on T cells

The phosphatidylserine-specific antibody bavituxi-mab (developed by Peregrine Pharmaceuticals) acts by dimerizing soluble β2-glycoprotein 1 converting it from a low to a high avidity binder of phosphatidylser-ine182 This allows an antitumour immune response to develop possibly by coating the tumour cells with anti-body or by converting apoptotic cell death to a more immunostimulatory type of cell death A Phase II trial of bavituximab in advanced NSCLC yielded positive results on progression-free survival and more impor-tantly improved overall survival from less than 6 months to 12 months183 leading the FDA to grant a fast track designation for bavituximab as a second-line therapy A Phase III trial of bavituximab as a second-line therapy for advanced NSCLC is underway Recent presentations showed synergy of phosphatidylserine-specific anti-body treatment with CTLA4-specific and PD1-specific

EctonucleotidasesEnzymes such as CD39 and CD73 that are expressed on the external cell surface and metabolize nucleotides

Fast track designationA US Food and Drug Administration (FDA) designation that expedites the FDA review process for a promising medication with the potential to address an unmet medical need for a serious or life-threatening condition with no adequate treatment or cure

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antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

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NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 19: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

antibodies in mouse models through effects on MDSC and lymphocyte activity184185 Bavituximab might also be beneficial when paired with a targeted therapy such as the VEGFR2-specific antibody ramucirumab (devel-oped by Eli Lilly and Company) which has reported positive Phase III trial data as second-line therapy in combination with docetaxel for NSCLC186

SIRPAndashCD47 In contrast to the phosphatidylserine sig-nal the signal-regulatory protein-α (SIRPA also known as CD172a)ndashCD47 system serves as a lsquodo not eat mersquo signal thereby preventing engulfment by macrophages Tumour cells are adept at hijacking the expression of CD47 to mask their abnormal proliferative phenotype Expression of CD47 on tumours shuts down macrophages via SIRPA and prevents phagocytosis187188 Notably cancer stem cells also utilize CD47 to escape the attention of mac-rophages189 Several companies are developing antibodies to CD47 or to SIRPA The approach is effective in models of human tumours in mouse models and there is a report of synergy with rituximab in a mouse model of non-Hodgkin lymphoma190 Inhibrx licensed a CD47-specific antibody to Celgene in 2014 Importantly this antibody is designed to not deplete red blood cells one of the on-tar-get toxicities associated with this pathway

The potential benefit of therapies with phosphatidyl-serine-specific mAbs or CD47-specific mAbs is that they should allow engagement of activated macrophages that will engulf tumour cells and present tumour antigens thereby stimulating an adaptive antitumour immune response This approach is similar to that of engaging dendritic cells for antigen presentation in tumour vac-cine strategies It is widely assumed that tumour vaccines will benefit from immune checkpoint combination ther-apy a hypothesis that has been supported by preclinical modelling (reviewed in REF 191)

ConclusionAn improved understanding of the immune response to cancer as well as patient selection and biomarker development has increased the number of patients who benefit from immunotherapies The goal of combination immunotherapies is to produce a durable antitumour response in patients who would not benefit from mono-therapy Accumulating evidence indicates that tumours that benefit from immunotherapy have an indolent underlying antitumour immune response Tumours may use multiple mechanisms of immune evasion which may vary between the treatment-naive setting and in response to the selective pressure of treatment192

The response to different immunotherapeutic combi-nations will probably depend on the individualrsquos immune milieu A lymphocyte infiltrate is a positive prognostic finding in most cancers193 Nevertheless lymphocytic infiltration is not incorporated into the standard prognos-tic classification The TNM classification is the standard staging system for solid tumours at diagnosis and incor-porates tumour burden (T) lymph node involvement (N) and metastasis (M) and was developed as a prognostic model for survival194 However within a given TNM stage there can be significant variability in patient outcome

which can partially be accounted for by the extent of lym-phocyte infiltration195 Because of the prognostic signifi-cance of the immune infiltrate in cancer an international task force was initiated to develop the Immunoscore as an adjunct to TNM staging196 The Immunoscore model is well developed in colon cancer and assessing the immune context in the primary colon tumour after resection improves the standard prognostic model in early-stage disease197 With the success of the immune checkpoint inhibitors developing a predictive Immunoscore for other tumour types will be even more clinically useful The combination of PD1+ CD8+ T cells and PDL1 expres-sion in the tumour may indicate an ongoing but exhausted antitumour immune response that is a prime candidate for immune checkpoint blockade

Although infiltrating lymphocytes are generally posi-tively prognostic in many tumour types the immune infil-trate is clearly insufficient to eliminate the tumour Lymph node-like structures have been described in tumours at the tumour margin and appear to be prognostic in mela-noma198 These tumours have the highest expression of a unique chemokine signature known to direct the organi-zation of lymphoid structuresThese results suggest that it is not merely the presence of tumour-infiltrating lym-phocytes but presentation of tumour antigen within a lymphoid structure that is important

In kidney cancer the opposite relationship was first described increased immune infiltrate and PDL1 expres-sion are a poor prognostic marker199 However further analysis including examination of lymphoid structure has shown that the combination of CD8+ T cell infiltrate lymphoid structure and low PDL1 expression is associated with positive prognosis in kidney cancer200

Unfortunately the identification of lymphoid struc-tures in tumours requires large specimens Ongoing studies in many tumours are focused on characterizing tumour-infiltrating lymphocytes including the over-expression of immune checkpoints such as PD1 LAG3 and TIM3 which are markers of exhaustion when highly expressed102109 In preclinical models combining immu-notherapies can overcome this exhausted phenotype101109

Infiltrating myeloid cells such as tumour-associated macrophages or MDSCs may express PDL1 and pro-duce inhibitory factors within the tumour that may be assayable in blood (for example VEGF) As our under-standing of predictors of response improves tissue- and blood-based analysis will be needed to characterize the immune environment and identify the best combination therapy for that milieu

We expect that integrating immunohistochemistry and gene expression signatures will improve predictive biomarker algorithms Incorporating gene expression signatures into predictive models may be an easier assay to translate into the clinic because gene expression signa-tures generate quantitative data on many genes simulta-neously which is in contrast to the labour- reagent- and analysis-intensive immunohistochemistry analysis

We hypothesize that distinct strategies of immune evasion exist before systemic therapy and after pro-gression on treatment be it chemotherapy or immu-notherapy Therefore the development of a system to

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 579

copy 2015 Macmillan Publishers Limited All rights reserved

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

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copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 20: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

predict response that considers not only PDL1 status and lymphocyte profile but also a broader picture of the immune milieu will be invaluable for directing thera-peutic combinations

Immune checkpoint blockade has been shown to be more likely to benefit patients with tumours that express PDL1 and have a CD8+ T cell immune infiltrate201 However some tumours have little or no immune infil-trate This may be a mechanism of immune evasion via immune ignorance wherein a tumour fails to have or present a tumour antigen recognized by the immune system Models incorporating both expression of immu-noinhibitors and the antigenicity of a patientrsquos neoanti-gens (derived by exome sequencing and in silico analysis of neoantigen presentation)3 may predict which patients are more likely to respond to immune checkpoint block-ade with or without other immunomodulators and which patients will need engineered effector cells (such as ex vivo expanded tumour-infiltrating lymphocytes or the engineered cellular therapies that utilize chimeric antigen receptors202 or cloned T cell receptors) or a per-sonalized vaccine3 Notably engineered T cells in vivo can begin to express exhaustion receptors and reduced activity that can be reversed by PD1 blockade203

There are many open questions in cancer immuno-therapy from basic immune biology to clinical analysis

While we learn more about the mechanisms of action of tumour immunotherapy and the development of resistance the field of immuno-oncology is also learn-ing the best way to study these agents in patients For example because 10ndash25 of patients who benefit from immune checkpoint blockade are not accounted for by the traditional response criteria (owing to inflammation giving the radiological appearance of tumour growth) the clinical benefit of immunotherapy may be greater than measured by standard response criteria204 As our understanding of the immune mechanisms of these agents increases and as prognostic and predictive algo-rithms improve this will help direct the development of combination therapies for clinical trials We predict that in the future a pathologist will examine a tumour biopsy for immune infiltrate and the expression of immunoin-hibitory markers on the tumour and tumour-infiltrating lymphocytes and use this information to direct an opti-mal regimen of checkpoint blockers and immune ago-nists The long-lasting effects of CTLA4 blockade for which most patients with melanoma alive at 3 years are still alive at follow-up at 10 years allow us to talk of a cure for cancer15 Now is an extraordinary time to be in the field and the next decade should provide the oppor-tunity to realize the potential of cancer immunotherapy to benefit more patients

1 Schreiber R D Old L J amp Smyth M J Cancer immunoediting integrating immunityrsquos roles in cancer suppression and promotion Science 331 1565ndash1570 (2011)

2 Fritsch E F et al HLA-binding properties of tumor neoepitopes in humans Cancer Immunol Res 2 522ndash529 (2014)

3 Gubin M M et al Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens Nature 515 577ndash581 (2014)Identifies tumour-specific mutant proteins (neoantigens) as the major T cell rejection targets following anti-PD1 andor anti-CTLA4 tumour immunotherapy in mice Vaccines incorporating these neoantigens can induce tumour rejection

4 Freeman G J et al Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation J Exp Med 192 1027ndash1034 (2000)Shows that PDL1 is a ligand for PD1 and inhibits T cell activation It also shows that PDL1 expression is upregulated by IFNγ

5 Blackburn S D et al Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection Nat Immunol 10 29ndash37 (2009)Shows that expression of multiple inhibitory receptors in addition to PD1 is associated with greater T cell exhaustion in a chronic viral infection model

6 Hodi F S et al Improved survival with ipilimumab in patients with metastatic melanoma N Engl J Med 363 711ndash723 (2010)A report of the Phase III trial showing a survival advantage with CTLA4-specific mAb treatment in advanced melanoma the results supported FDA approval of the first immune checkpoint inhibitor

7 Powles T et al MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer Nature 515 558ndash562 (2014)Although not the first published report of efficacy of therapeutically targeting PDL1 this study demonstrates a 43 response rate in metastatic bladder cancer a good safety profile and a threefold increase in response rates in tumours that express PDL1 on the tumour-infiltrating immune cells

8 Hamid O et al Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma N Engl J Med 369 134ndash144 (2013)

9 Brahmer J R et al Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors safety clinical activity pharmacodynamics and immunologic correlates J Clin Oncol 28 3167ndash3175 (2010)This first-in-human trial of a PD1 blocker shows that the therapy is well tolerated and produces a partial response in a patient with melanoma and in a patient with renal cell carcinoma and a complete response in a patient with colon adenocarcinoma

10 Herbst R S et al Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients Nature 515 563ndash567 (2014)

11 Garon E B et al Pembrolizumab for the treatment of non-small-cell lung cancer N Engl J Med 372 2018ndash2028 (2015)

12 Le D T et al PD-1 blockade in tumors with mismatch-repair deficiency N Engl J Med httpdxdoiorg101056nejmoa1500596 (2015)

13 Robert C et al Pembrolizumab versus ipilimumab in advanced melanoma N Engl J Med httpdxdoiorg101056nejmoa1503093 (2015)

14 Larkin J et al Combined nivolumab and ipilimumab or monotherapy in untreated melanoma N Engl J Med httpdxdoiorg101056nejmoa1504030 (2015)

15 Schadendorf D et al Pooled analysis of long-term survival data from Phase II and Phase III trials of ipilimumab in unresectable or metastatic melanoma J Clin Oncol 33 1889ndash1894 (2015) Pooled data from multiple clinical trials of ipilimumab with follow-up for up to 10 years (4846 patients) demonstrate that the survival curve begins to plateau in year three indicating durable ongoing responses

16 Wolchok J D et al Nivolumab plus ipilimumab in advanced melanoma N Engl J Med 369 122ndash133 (2013)The first Phase I trial of combined CTLA4 and PD1 blockade demonstrates that this combination is highly effective but highly toxic

17 Postow M A et al Nivolumab and ipilimumab versus ipilimumab in untreated melanoma N Engl J Med 372 2006ndash2017 (2015)

The first randomized Phase II trial to compare the efficacy and toxicity of combination versus monotherapy with immune checkpoint inhibitors It reports 61 response rates with nivolumab plus ipilimumab versus 11 with ipilimumab alone however 54 of patients on the combination had grade 3 or 4 adverse events

18 Jenkins M K amp Schwartz R H Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo J Exp Med 165 302ndash319 (1987)

19 Suntharalingam G et al Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412 N Engl J Med 355 1018ndash1028 (2006)

20 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) and ipilimumab in first-line NSCLC interim phase I results J Clin Oncol 32 (Suppl) 8023 (2014)

21 Antonia S J et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with platinum-based doublet chemotherapy (PT-DC) in advanced non-small cell lung cancer (NSCLC) J Clin Oncol 32 (Suppl) 8113 (2014)

22 Amin A et al Nivolumab (anti-PD-1 BMS-936558 ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 5010 (2014)

23 Hammers H et al Phase I study of nivolumab in combination with ipilimumab in metastatic renal cell carcinoma (mRCC) J Clin Oncol 32 (Suppl) 4504 (2014)

24 Hamid O S et al Clinical activity safety and biomarkers of MPDL3280A an engineered PD-L1 antibody in patients with locally advanced or metastatic melanoma (mM) J Clin Oncol 31 (Suppl) 9010 (2013)

25 Postow M A et al Immunologic correlates of the abscopal effect in a patient with melanoma N Engl J Med 366 925ndash931 (2012)

26 Antonia S et al Phase Ib study of MEDI4736 a programmed cell death ligand-1 (PD-L1) antibody in combination with tremelimumab a cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) antibody in patients (pts) with advanced NSCLC J Clin Oncol 33 (Suppl) 3014 (2015)

R E V I E W S

580 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 21: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

27 Parry R V et al CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms Mol Cell Biol 25 9543ndash9553 (2005)

28 Korman A et al Activity of anti-PD-1 in murine tumor models role of ldquohostrdquo PD-L1 and synergistic effect of anti-PD-1 and anti-CTLA-4 J Immunol 178 (Suppl) S82 (2007)First report to demonstrate the synergistic effect of blocking both PD1 and CTLA4 in mouse tumour models the results formed the basis of the development of this combination in patients

29 Freeman G J amp Sharpe A H A new therapeutic strategy for malaria targeting T cell exhaustion Nat Immunol 13 113ndash115 (2012)

30 Robert C et al Nivolumab in previously untreated melanoma without BRAF mutation N Engl J Med 372 320ndash330 (2015)

31 Dovedi S J et al Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade Cancer Res 74 5458ndash5468 (2014)

32 Keir M E Butte M J Freeman G J amp Sharpe A H PD-1 and its ligands in tolerance and immunity Annu Rev Immunol 26 677ndash704 (2008)

33 Postow M A et al Pharmacodynamic effect of ipilimumab on absolute lymphocyte count (ALC) and association with overall survival in patients with advanced melanoma J Clin Oncol 31 (Suppl) 9052 (2013)

34 Le D T amp Jaffee E M Regulatory T-cell modulation using cyclophosphamide in vaccine approaches a current perspective Cancer Res 72 3439ndash3444 (2012)

35 Vincent J et al 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity Cancer Res 70 3052ndash3061 (2010)

36 Sevko A et al Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model J Immunol 190 2464ndash2471 (2013)

37 Hannani D et al Prerequisites for the antitumor vaccine-like effect of chemotherapy and radiotherapy Cancer J 17 351ndash358 (2011)

38 Kim K et al Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells Proc Natl Acad Sci USA 111 11774ndash11779 (2014)

39 Papa M Z Vetto J T Ettinghausen S E Mule J J amp Rosenberg S A Effect of corticosteroid on the antitumor activity of lymphokine-activated killer cells and interleukin 2 in mice Cancer Res 46 5618ndash5623 (1986)

40 Mier J W et al Inhibition of interleukin-2-induced tumor necrosis factor release by dexamethasone prevention of an acquired neutrophil chemotaxis defect and differential suppression of interleukin-2-associated side effects Blood 76 1933ndash1940 (1990)

41 Vetto J T Papa M Z Lotze M T Chang A E amp Rosenberg S A Reduction of toxicity of interleukin-2 and lymphokine-activated killer cells in humans by the administration of corticosteroids J Clin Oncol 5 496ndash503 (1987)

42 Wolchok J et al Efficacy and safety results from a Phase III trial of nivolumab (NIVO) alone or combined with ipilimumab (IPI) versus IPI alone in treatment-naive patients (pts) with advanced melanoma (MEL) (CheckMate 067) J Clin Oncol 33 (Suppl) LBA1 (2015)

43 Donia M et al BRAF inhibition improves tumor recognition by the immune system potential implications for combinatorial therapies against melanoma involving adoptive T-cell transfer Oncoimmunology 1 1476ndash1483 (2012)

44 Frederick D T et al BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma Clin Cancer Res 19 1225ndash1231 (2013)

45 Jiang X Zhou J Giobbie-Hurder A Wargo J amp Hodi F S The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition Clin Cancer Res 19 598ndash609 (2013)

46 Cooper Z A et al Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade Cancer Immunol Res 2 643ndash654 (2014)

47 Ribas A Hodi F S Callahan M Konto C amp Wolchok J Hepatotoxicity with combination of

vemurafenib and ipilimumab N Engl J Med 368 1365ndash1366 (2013)

48 Ribas A et al Phase I study combining anti-PD-L1 (MEDI4736) with BRAF (dabrafenib) andor MEK (trametinib) inhibitors in advanced melanoma J Clin Oncol 33 (Suppl) 3003 (2015)

49 Talmadge J E amp Gabrilovich D I History of myeloid-derived suppressor cells Nat Rev Cancer 13 739ndash752 (2013)

50 Finke J et al MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy Int Immunopharmacol 11 856ndash861 (2011)

51 Bhatt R S et al Renal cancer resistance to antiangiogenic therapy is delayed by restoration of angiostatic signaling Mol Cancer Ther 9 2793ndash2802 (2010)

52 Salven P Orpana A Teerenhovi L amp Joensuu H Simultaneous elevation in the serum concentrations of the angiogenic growth factors VEGF and bFGF is an independent predictor of poor prognosis in non-Hodgkin lymphoma a single-institution study of 200 patients Blood 96 3712ndash3718 (2000)

53 Zhan P Qian Q amp Yu L K Serum VEGF level is associated with the outcome of patients with hepatocellular carcinoma a meta-analysis Hepatobiliary Surg Nutr 2 209ndash215 (2013)

54 Yuan J et al Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab Cancer Immunol Res 2 127ndash132 (2014)

55 Ohm J E amp Carbone D P VEGF as a mediator of tumor-associated immunodeficiency Immunol Res 23 263ndash272 (2001)

56 Gabrilovich D I Ishida T Nadaf S Ohm J E amp Carbone D P Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function Clin Cancer Res 5 2963ndash2970 (1999)

57 Lieu C et al Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) andor chemotherapy (chemo) in patients (pts) with locally advanced or metastatic solid tumors Ann Oncol 25 (Suppl 4) iv361ndashiv372 (2014)This is the first report of the combination of a PDL1-specific mAb and a VEGF-specific mAb (bevacizumab) in patients which was highly tolerable in patients

58 Ellis L M The role of neuropilins in cancer Mol Cancer Ther 5 1099ndash1107 (2006)

59 Chaudhary B Khaled Y S Ammori B J amp Elkord E Neuropilin 1 function and therapeutic potential in cancer Cancer Immunol Immunother 63 81ndash99 (2014)

60 Papatriantafyllou M T cells neuropilin 1 mdash distinguishing TReg cell subsets Nat Rev Immunol 12 746 (2012)

61 Grandclement C amp Borg C Neuropilins a new target for cancer therapy Cancers (Basel) 3 1899ndash1928 (2011)

62 Weekes C D et al A Phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors Invest New Drugs 32 653ndash660 (2014)

63 Patnaik A et al Phase Ib study evaluating MNRP1685A a fully human anti-NRP1 monoclonal antibody in combination with bevacizumab and paclitaxel in patients with advanced solid tumors Cancer Chemother Pharmacol 73 951ndash960 (2014)

64 Delgoffe G M et al Stability and function of regulatory T cells is maintained by a neuropilin-1-semaphorin-4a axis Nature 501 252ndash256 (2013)

65 Vilcek J amp Feldmann M Historical review cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201ndash209 (2004)

66 Schaer D A Hirschhorn-Cymerman D amp Wolchok J D Targeting tumor-necrosis factor receptor pathways for tumor immunotherapy J Immunother Cancer 2 7 (2014)

67 Kwon B S et al Immune responses in 4-1BB (CD137)-deficient mice J Immunol 168 5483ndash5490 (2002)

68 Chester C et al Biomarker characterization using mass cytometry in a phase 1 trial of urelumab (BMS-663513) in subjects with advanced solid tumors and relapsedrefractory B-cell non-Hodgkin lymphoma J Clin Oncol 32 (Suppl) 3017 (2014)

69 Hwu W Targeted therapy for metastatic melanoma from bench to bedside HemOnc Today [online] httpwwwhealiocomhematology-oncologymelanoma-skin-cancernewsprinthemonc-today7B77e71a11-1fd1-4193-a2f7-7c50c6121c1f7Dtargeted-

therapy-for-metastatic-melanoma-from-bench-to-bedside (2010)

70 Segal N H et al A phase 1 study of PF-05082566 (anti-4-1BB) in patients with advanced cancer J Clin Oncol 32 (Suppl) 3007 (2014)

71 Gopal A et al A phase I study of PF-05082566 (anti-4-1BB) + rituximab in patients with CD20+ NHL J Clin Oncol 33 (Suppl) 3004 (2015)

72 Kohrt H E et al Targeting CD137 enhances the efficacy of cetuximab J Clin Invest 124 2668ndash2682 (2014)

73 Ye Q et al CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor Clin Cancer Res 20 44ndash55 (2014)Demonstrates that in the tumour microenvironment tumour-reactive T cells express the TNFR 4-1BB thus bolstering the hypothesis that 4-1BB-specific agonist antibodies will have a synergistic effect with other immune checkpoint therapeutics

74 Bulliard Y et al OX40 engagement depletes intratumoral Tregs via activating FcγRs leading to antitumor efficacy Immunol Cell Biol 92 475ndash480 (2014)

75 Guo Z et al PD-1 blockade and OX40 triggering synergistically protects against tumor growth in a murine model of ovarian cancer PLoS ONE 9 e89350 (2014)

76 Guo Z et al Combined TIM-3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer J Transl Med 11 215 (2013)

77 Adler A J amp Vella A T Betting on improved cancer immunotherapy by doubling down on CD134 and CD137 co-stimulation Oncoimmunology 2 e22837 (2013)

78 Wei H et al Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin PLoS ONE 8 e84927 (2013)

79 Kovacsovics-Bankowski M et al Phase III clinical trial of anti-OX40 radiation and cyclophosphamide in patients with prostate cancer immunological analysis J Immunother Cancer 1 255 (2013)

80 Curti B D et al OX40 is a potent immune-stimulating target in late-stage cancer patients Cancer Res 73 7189ndash7198 (2013)

81 Cohen A D et al Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation PLoS ONE 5 e10436 (2010)

82 Lu L et al Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs J Transl Med 12 36 (2014)

83 Grewal I S CD70 as a therapeutic target in human malignancies Expert Opin Ther Targets 12 341ndash351 (2008)

84 Ansell S M et al Phase I evaluation of an agonist anti-CD27 human antibody (CDX-1127) in patients with advanced hematologic malignancies J Clin Oncol 32 (Suppl) 3034 (2014)

85 Infante J R et al Immunologic activity of an activating anti-CD27 antibody (CDX-1127) in patients (pts) with solid tumors J Clin Oncol 32 (Suppl) 3027 (2014)

86 Twohig J P et al The death receptor 3TL1A pathway is essential for efficient development of antiviral CD4+ and CD8+ T-cell immunity FASEB J 26 3575ndash3586 (2012)

87 Bossen C et al Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human J Biol Chem 281 13964ndash13971 (2006)

88 Meylan F et al The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases Immunity 29 79ndash89 (2008)

89 Slebioda T J et al Triggering of TNFRSF25 promotes CD8+ T-cell responses and anti-tumor immunity Eur J Immunol 41 2606ndash2611 (2011)

90 Schreiber T H amp Podack E R Immunobiology of TNFSF15 and TNFRSF25 Immunol Res 57 3ndash11 (2013)

91 Schreiber T H Wolf D Bodero M Gonzalez L amp Podack E R T cell costimulation by TNFR superfamily (TNFRSF)4 and TNFRSF25 in the context of vaccination J Immunol 189 3311ndash3318 (2012)

92 Bodmer J L Schneider P amp Tschopp J The molecular architecture of the TNF superfamily Trends Biochem Sci 27 19ndash26 (2002)

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 581

copy 2015 Macmillan Publishers Limited All rights reserved

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 22: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

93 Bajor D et al Combination of agonistic CD40 monoclonal antibody CP-870893 and anti-CTLA-4 antibody tremelimumab in patients with metastatic melanoma American Association for Cancer Research Annual Meeting [online] httpwwwabstractsonlinecomPlanViewAbstractaspxmID=3682ampsKey= b6e2d928-3a12-44b7-9f8a-3a69ffde9741ampcKey= b51efb80-7710-4f0e-949b-623213c6ff47ampmKey =19573a54-ae8f-4e00-9c23-bd6d62268424 (2015)

94 Zippelius A Schreiner J Herzig P amp Muller P Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment Cancer Immunol Res 3 236ndash244 (2015)

95 Medina-Echeverz J et al Systemic agonistic anti-CD40 treatment of tumor-bearing mice modulates hepatic myeloid-suppressive cells and causes immune-mediated liver damage Cancer Immunol Res 3 557ndash566 (2015)

96 Cai G et al CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator Nat Immunol 9 176ndash185 (2008)

97 Derre L et al BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination J Clin Invest 120 157ndash167 (2010)

98 Pasero C amp Olive D Interfering with coinhibitory molecules BTLAHVEM as new targets to enhance anti-tumor immunity Immunol Lett 151 71ndash75 (2013)

99 Watanabe N et al BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1 Nat Immunol 4 670ndash679 (2003)

100 Huang C T et al Role of LAG-3 in regulatory T cells Immunity 21 503ndash513 (2004)

101 Woo S R et al Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape Cancer Res 72 917ndash927 (2012)The combination of LAG3 and PD1 blockade in a mouse tumour model is more effective than either alone prompting the development of this combination in an ongoing clinical trial

102 Matsuzaki J et al Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer Proc Natl Acad Sci USA 107 7875ndash7880 (2010)

103 Wang-Gillam A et al A Phase I study of IMP321 and gemcitabine as the front-line therapy in patients with advanced pancreatic adenocarcinoma Invest New Drugs 31 707ndash713 (2013)

104 Brignone C et al First-line chemoimmunotherapy in metastatic breast carcinoma combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity J Transl Med 8 71 (2010)

105 Brignone C Escudier B Grygar C Marcu M amp Triebel F A phase I pharmacokinetic and biological correlative study of IMP321 a novel MHC class II agonist in patients with advanced renal cell carcinoma Clin Cancer Res 15 6225ndash6231 (2009)

106 McIntire J J et al Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family Nat Immunol 2 1109ndash1116 (2001)

107 Freeman G J Casasnovas J M Umetsu D T amp DeKruyff R H TIM genes a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity Immunol Rev 235 172ndash189 (2010)

108 Jin H T et al Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection Proc Natl Acad Sci USA 107 14733ndash14738 (2010)

109 Sakuishi K et al Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity J Exp Med 207 2187ndash2194 (2010)Promising results from the combination of TIM3 and PD1 blockade in mouse tumours support the development of this combination in patients

110 Zhu C et al The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity Nat Immunol 6 1245ndash1252 (2005)

111 Huang Y H et al CEACAM1 regulates TIM-3-mediated tolerance and exhaustion Nature 517 386ndash390 (2015)

112 DeKruyff R H et al T celltransmembrane Ig and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells J Immunol 184 1918ndash1930 (2010)

113 Sakuishi K Jayaraman P Behar S M Anderson A C amp Kuchroo V K Emerging Tim-3

functions in antimicrobial and tumor immunity Trends Immunol 32 345ndash349 (2011)

114 Madireddi S et al Galectin-9 controls the therapeutic activity of 4-1BB-targeting antibodies J Exp Med 211 1433ndash1448 (2014)

115 Gleason M K et al Tim-3 is an inducible human natural killer cell receptor that enhances interferon γ production in response to galectin-9 Blood 119 3064ndash3072 (2012)

116 Pillai S Netravali I A Cariappa A amp Mattoo H Siglecs and immune regulation Annu Rev Immunol 30 357ndash392 (2012)

117 Jandus C et al Interactions between Siglec-79 receptors and ligands influence NK cell-dependent tumor immunosurveillance J Clin Invest 124 1810ndash1820 (2014)

118 Laubli H et al Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer Proc Natl Acad Sci USA 111 14211ndash14216 (2014)

119 Takamiya R Ohtsubo K Takamatsu S Taniguchi N amp Angata T The interaction between Siglec-15 and tumor-associated sialyl-Tn antigen enhances TGF-β secretion from monocytesmacrophages through the DAP12-Syk pathway Glycobiology 23 178ndash187 (2013)

120 Pardoll D M The blockade of immune checkpoints in cancer immunotherapy Nat Rev Cancer 12 252ndash264 (2012)

121 Chen L amp Flies D B Molecular mechanisms of T cell co-stimulation and co-inhibition Nat Rev Immunol 13 227ndash242 (2013)

122 Zhao R et al HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function Proc Natl Acad Sci USA 110 9879ndash9884 (2013)

123 Sharpe A H Mechanisms of costimulation Immunol Rev 229 5ndash11 (2009)

124 Capece D Verzella D Fischietti M Zazzeroni F amp Alesse E Targeting costimulatory molecules to improve antitumor immunity J Biomed Biotechnol 2012 926321 (2012)

125 Ng Tang D et al Increased frequency of ICOS+ CD4 T cells as a pharmacodynamic biomarker for anti-CTLA-4 therapy Cancer Immunol Res 1 229ndash234 (2013)

126 Fan X Quezada S A Sepulveda M A Sharma P amp Allison J P Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy J Exp Med 211 715ndash725 (2014)

127 Yap E H Rosche T Almo S amp Fiser A Functional clustering of immunoglobulin superfamily proteins with proteinndashprotein interaction information calibrated hidden Markov model sequence profiles J Mol Biol 426 945ndash961 (2014)

128 Le Mercier I et al VISTA regulates the development of protective antitumor immunity Cancer Res 74 1933ndash1944 (2014)

129 Janakiram M et al Expression clinical significance and receptor identification of the newest B7 family member HHLA2 protein Clin Cancer Res 21 2359ndash2366 (2015)

130 Zhu Y et al B7-H5 costimulates human T cells via CD28H Nat Commun 4 2043 (2013)

131 Nguyen T Liu X K Zhang Y amp Dong C BTNL2 a butyrophilin-like molecule that functions to inhibit T cell activation J Immunol 176 7354ndash7360 (2006)

132 Valentonyte R et al Sarcoidosis is associated with a truncating splice site mutation in BTNL2 Nat Genet 37 357ndash364 (2005)

133 Arnett H A amp Viney J L Immune modulation by butyrophilins Nat Rev Immunol 14 559ndash569 (2014)

134 Fitzgerald L M et al Germline missense variants in the BTNL2 gene are associated with prostate cancer susceptibility Cancer Epidemiol Biomarkers Prev 22 1520ndash1528 (2013)

135 Vijai J et al A genome-wide association study suggests evidence of variants at 6p2132 associated with marginal zone lymphoma Cancer Res 74 (Suppl 19) 5071 (2014)

136 Abeler-Dorner L Swamy M Williams G Hayday A C amp Bas A Butyrophilins an emerging family of immune regulators Trends Immunol 33 34ndash41 (2012)

137 Rubinstein R Ramagopal U A Nathenson S G Almo S C amp Fiser A Functional classification of immune regulatory proteins Structure 21 766ndash776 (2013)

138 de Andrade L F Smyth M J amp Martinet L DNAM-1 control of natural killer cells functions

through nectin and nectin-like proteins Immunol Cell Biol 92 237ndash244 (2014)

139 Sema Kurtulus S et al Mechanisms of TIGIT-driven immune suppression in cancer J Immunother Cancer 2 (Suppl 3) O13 (2014)

140 Chauvin J M et al TIGIT and PD-1 impair tumor antigen-specific CD8+ T cells in melanoma patients J Clin Invest 125 2046ndash2058 (2015)

141 Johnston R J et al The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function Cancer Cell 26 923ndash937 (2014)TIGIT is identified as an inhibitory receptor mediating exhaustion on CD8+ T cells the combination of TIGIT and PDL1 blockade is more effective than either alone in preclinical models

142 Guillerey C et al Immunosurveillance and therapy of multiple myeloma are CD226 dependent J Clin Invest 125 2077ndash2089 (2015)

143 Li A H et al Analysis of loss-of-function variants and 20 risk factor phenotypes in 8554 individuals identifies loci influencing chronic disease Nat Genet 47 640ndash642 (2015)

144 Beano A et al Correlation between NK function and response to trastuzumab in metastatic breast cancer patients J Transl Med 6 25 (2008)Elucidates the critical role of natural killer cells in mediating the activity of trastuzumab in patients with breast cancer

145 Vey N et al A Phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission Blood 120 4317ndash4323 (2012)

146 Raulet D H Roles of the NKG2D immunoreceptor and its ligands Nat Rev Immunol 3 781ndash790 (2003)

147 Spear P Wu M R Sentman M L amp Sentman C L NKG2D ligands as therapeutic targets Cancer Immun 13 8 (2013)

148 Farnault L Sanchez C Baier C Le Treut T amp Costello R T Hematological malignancies escape from NK cell innate immune surveillance mechanisms and therapeutic implications Clin Dev Immunol 2012 421702 (2012)

149 Bonnafous C et al Targeting MICA with therapeutic antibodies for the treatment of cancer (Poster) J Immunother Cancer 1 (Suppl 1) P41 (2013)

150 Wada H Matsumoto N Maenaka K Suzuki K amp Yamamoto K The inhibitory NK cell receptor CD94NKG2A and the activating receptor CD94NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues Eur J Immunol 34 81ndash90 (2004)

151 Chretien A S et al Cancer-induced alterations of NK-mediated target recognition current and investigational pharmacological strategies aiming at restoring NK-mediated anti-tumor activity Front Immunol 5 122 (2014)

152 Sheu J amp Shih le-M HLA-G and immune evasion in cancer cells J Formos Med Assoc 109 248ndash257 (2010)

153 Alegre E et al Some basic aspects of HLA-G biology J Immunol Res 2014 657625 (2014)

154 Gonzaacutelez A et al The immunosuppressive molecule HLA-G and its clinical implications Crit Rev Clin Lab Sci 49 63ndash84 (2012)

155 Stark S amp Watzl C 2B4 (CD244) NTB-A and CRACC (CS1) stimulate cytotoxicity but no proliferation in human NK cells Int Immunol 18 241ndash247 (2006)

156 Valiante N M amp Trinchieri G Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes J Exp Med 178 1397ndash1406 (1993)

157 Ljunggren H G amp Malmberg K J Prospects for the use of NK cells in immunotherapy of human cancer Nat Rev Immunol 7 329ndash339 (2007)

158 Munn D H Blocking IDO activity to enhance anti-tumor immunity Front Biosci (Elite Ed) 4 734ndash745 (2012)This review highlights immune suppression mediated by IDO in both the tumour and its draining lymph nodes

159 Gibney G T et al Preliminary results from a phase 12 study of INCB024360 combined with ipilimumab (ipi) in patients (pts) with melanoma J Clin Oncol 32 (Suppl) 3010 (2014)

160 Soliman H H et al A first in man phase I trial of the oral immunomodulator indoximod combined with docetaxel in patients with metastatic solid tumors Oncotarget 5 8136ndash8146 (2014)

161 [No authors listed] NewLink Genetics announces exclusive worldwide licensing agreement for development of NLG919 an IDO inhibitor in Phase 1

R E V I E W S

582 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

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203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 23: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

and research collaboration for the discovery of next generation IDOTDO inhibitors NewLink Genetics [online] httpinvestorslinkpcomreleasedetailcfmreleaseid=876909 (2014)

162 [No authors listed] Bristol-Myers Squibb to expand its immuno-oncology pipeline with agreement to acquire Flexus Biosciences Inc Bristol‑Myers Squibb [online] httpnewsbmscompress-releaserd-newsbristol-myers-squibb-expand-its-immuno-oncology-pipeline-agreement-acquire-fle (2015)

163 Margadant C amp Sonnenberg A Integrin-TGF-β crosstalk in fibrosis cancer and wound healing EMBO Rep 11 97ndash105 (2010)

164 Gueorguieva I et al Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokineticpharmacodynamic model Br J Clin Pharmacol 77 796ndash807 (2014)

165 Bhola N E et al TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer J Clin Invest 123 1348ndash1358 (2013)Shows that TGFszlig-dependent signals underlie resistance to chemotherapy via multiple mechanisms including blunting the antitumour immune response

166 Bedi A et al Inhibition of TGF-β enhances the in vivo antitumor efficacy of EGF receptor-targeted therapy Mol Cancer Ther 11 2429ndash2439 (2012)

167 Hanks B A Holtzhausen A Evans K Heid M amp Blobe G C Combinatorial TGF-β signaling blockade and anti-CTLA-4 antibody immunotherapy in a murine BRAFV600E-PTENminusminus transgenic model of melanoma J Clin Oncol 32 (Suppl) 3011 (2014)

168 Wilson W R amp Hay M P Targeting hypoxia in cancer therapy Nat Rev Cancer 11 393ndash410 (2011)

169 Ohta A et al A2A adenosine receptor protects tumors from antitumor T cells Proc Natl Acad Sci USA 103 13132ndash13137 (2006)

170 Cekic C amp Linden J Adenosine A2A receptors intrinsically regulate CD8+ T cells in the tumor microenvironment Cancer Res 74 7239ndash7249 (2014)

171 Jones G B amp Yuan G Towards next generation adenosine A2A receptor antagonists Curr Med Chem 21 3918ndash3935 (2014)

172 Yegutkin G G Nucleotide- and nucleoside-converting ectoenzymes important modulators of purinergic signalling cascade Biochim Biophys Acta 1783 673ndash694 (2008)

173 Mandapathil M et al Generation and accumulation of immunosuppressive adenosine by human CD4+CD25highFOXP3+ regulatory T cells J Biol Chem 285 7176ndash7186 (2010)

174 Iannone R Miele L Maiolino P Pinto A amp Morello S Adenosine limits the therapeutic effectiveness of anti-CTLA4 mAb in a mouse melanoma model Am J Cancer Res 4 172ndash181 (2014)

175 Allard B Pommey S Smyth M J amp Stagg J Targeting CD73 enhances the antitumor activity of anti-PD-1 and anti-CTLA-4 mAbs Clin Cancer Res 19 5626ndash5635 (2013)Shows that blocking CD73 improves the effect of both PD1 and CTLA4 blockade and preferentially synergizes with PD1 blockade because activation of the adenosine receptor enhances PD1 expression

176 Hatfield S M et al Immunological mechanisms of the antitumor effects of supplemental oxygenation Sci Transl Med 7 277ra30 (2015)

177 Cekic C Day Y J Sag D amp Linden J Myeloid expression of adenosine A2A receptor suppresses T and NK cell responses in the solid tumor microenvironment Cancer Res 74 7250ndash7259 (2014)

178 Morello S amp Miele L Targeting the adenosine A2b receptor in the tumor microenvironment overcomes local immunosuppression by myeloid-derived suppressor cells OncoImmunology 3 e27989 (2014)

179 Hoskin D W Mader J S Furlong S J Conrad D M amp Blay J Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (Review) Int J Oncol 32 527ndash535 (2008)

180 Srivastava M K et al Myeloid suppressor cell depletion augments antitumor activity in lung cancer PLoS ONE 7 e40677 (2012)

181 Feig C et al Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer Proc Natl Acad Sci USA 110 20212ndash20217 (2013)

182 Luster T A et al Plasma protein β-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells J Biol Chem 281 29863ndash29871 (2006)

183 Shtivelband M et al Randomized blinded placebo-controlled phase II trial of docetaxel and bavituximab as second-line therapy in locally advanced or metastatic non-squamous non-small cell lung cancer J Clin Oncol 31 (Suppl) 8095 (2013)

184 Yopp A et al Antibody-mediated blockade of phosphatidylserine enhances the antitumor activity of targeted therapy and immune checkpoint inhibitors by affecting myeloid and lymphocyte populations in the tumor microenvironment J Immunother Cancer 2 (Suppl 3) P266 (2014)Blocking phosphotidylserine a ligand of the co-inhibitory receptor TIM3 improves the efficacy of PD1 and CTLA4 tumour immunotherapy

185 Huang X et al Antibody-mediated phosphatidylserine blockade significantly enhances the efficacy of downstream immune checkpoint inhibition in K1735 mouse melanoma J Immunother Cancer 2 (Suppl 3) P205 (2014)

186 Garon E B et al Ramucirumab plus docetaxel versus placebo plus docetaxel for second-line treatment of stage IV non-small-cell lung cancer after disease progression on platinum-based therapy (REVEL) a multicentre double-blind randomised phase 3 trial Lancet 384 665ndash673 (2014)

187 Tseng D et al Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response Proc Natl Acad Sci USA 110 11103ndash11108 (2013)Targeting CD47 increases phagocytosis enhances antitumour responses and synergizes with many antitumour mAbs in vivo

188 Weiskopf K et al Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies Science 341 88ndash91 (2013)

189 Jaiswal S et al CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis Cell 138 271ndash285 (2009)

190 Chao M P et al Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma Cell 142 699ndash713 (2010)

191 Melero I et al Therapeutic vaccines for cancer an overview of clinical trials Nat Rev Clin Oncol 11 509ndash524 (2014)

192 Taube J M et al Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape Sci Transl Med 4 127ra37 (2012)Describes the feedback loop whereby tumours dominantly evade the immune response by expressing PDL1 in response to IFNγ produced by activated T cells at the lymphocyte-rich tumour margin

193 Jass J R Lymphocytic infiltration and survival in rectal cancer J Clin Pathol 39 585ndash589 (1986)

194 Sobin L amp Wittekind C TNM Classification of Malignant Tumors 2nd edn (Wiley 2002)

195 Mlecnik B Bindea G Pages F amp Galon J Tumor immunosurveillance in human cancers Cancer Metastasis Rev 30 5ndash12 (2011)

196 Galon J et al Cancer classification using the Immunoscore a worldwide task force J Transl Med 10 205 (2012)

197 Angell H amp Galon J From the immune contexture to the Immunoscore the role of prognostic and predictive immune markers in cancer Curr Opin Immunol 25 261ndash267 (2013)

198 Messina J L et al 12-chemokine gene signature identifies lymph node-like structures in melanoma potential for patient selection for immunotherapy Sci Rep 2 765 (2012)

199 Thompson R H et al Costimulatory B7-H1 in renal cell carcinoma patients indicator of tumor aggressiveness and potential therapeutic target Proc Natl Acad Sci USA 101 17174ndash17179 (2004)The first article to show that PDL1 expression on tumour cells and on infiltrating lymphocytes in kidney cancer is associated with worse prognosis and increased cancer-specific death

200 Giraldo N A et al Orchestration and prognostic significance of immune checkpoints in the microenvironment of primary and metastatic renal cell cancer Clin Cancer Res httpdxdoiorg1011581078-0432CCR-14-2926 (2015)

201 Tumeh P C et al PD-1 blockade induces responses by inhibiting adaptive immune resistance Nature 515 568ndash571 (2014)

Shows that tumours that have high CD8 infiltrate and PDL1 expression on the tumour and at the tumour margin are more likely to respond to PD1 immunotherapy

202 Porter D L Levine B L Kalos M Bagg A amp June C H Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia N Engl JMed 365 725ndash733 (2011)

203 John L B et al Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells Clin Cancer Res 19 5636ndash5646 (2013)Shows that chimeric antigen receptor T cells can express PD1 be susceptible to PD1-mediated inhibition of T cell activation and that PD1 blockade can enhance the activity of chimeric antigen receptor T cells in mouse models

204 Wolchok J D et al Guidelines for the evaluation of immune therapy activity in solid tumors immune-related response criteria Clin Cancer Res 15 7412ndash7420 (2009)

205 Nordlund J J et al Vitiligo in patients with metastatic melanoma a good prognostic sign J Am Acad Dermatol 9 689ndash696 (1983)

206 Bystryn J C Rigel D Friedman R J amp Kopf A Prognostic significance of hypopigmentation in malignant melanoma Arch Dermatol 123 1053ndash1055 (1987)

207 Scalzo S et al Primary hypothyroidism associated with interleukin-2 and interferon α-2 therapy of melanoma and renal carcinoma Eur J Cancer 26 1152ndash1156 (1990)

208 Becker J C Winkler B Klingert S amp Brocker E B Antiphospholipid syndrome associated with immunotherapy for patients with melanoma Cancer 73 1621ndash1624 (1994)

209 Rosenberg S A amp White D E Vitiligo in patients with melanoma normal tissue antigens can be targets for cancer immunotherapy J Immunother Emphasis Tumor Immunol 19 81ndash84 (1996)

210 Atkins M B et al Hypothyroidism after treatment with interleukin-2 and lymphokine-activated killer cells N Engl J Med 318 1557ndash1563 (1988)

211 Attia P et al Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4 J Clin Oncol 23 6043ndash6053 (2005)

212 Chapman P B et al Improved survival with vemurafenib in melanoma with BRAF V600E mutation N Engl J Med 364 2507ndash2516 (2011)

213 Beck K E et al Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4 J Clin Oncol 24 2283ndash2289 (2006)

214 Kyi C Carvajal R D Wolchok J D amp Postow M A Ipilimumab in patients with melanoma and autoimmune disease J Immunother Cancer 2 35 (2014)

215 Hurwitz A A et al Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade Cancer Res 60 2444ndash2448 (2000)

216 Hodi F S et al Ipilimumab plus sargramostim versus ipilimumab alone for treatment of metastatic melanoma a randomized clinical trial JAMA 312 1744ndash1753 (2014)Treatment with ipilimumab plus sargramostim (a granulocytendashmacrophage colony-stimulating factor) versus ipilimumab alone results in lower toxicity and modestly better overall survival

217 Jinushi M et al MFG-E8-mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF J Clin Invest 117 1902ndash1913 (2007)

218 Rini B I et al Phase 1 dose-escalation trial of tremelimumab plus sunitinib in patients with metastatic renal cell carcinoma Cancer 117 758ndash767 (2011)

219 Maker A V et al Tumor regression and autoimmunity in patients treated with cytotoxic T lymphocyte-associated antigen 4 blockade and interleukin 2 a phase III study Ann Surg Oncol 12 1005ndash1016 (2005)

220 Robert C et al Ipilimumab plus dacarbazine for previously untreated metastatic melanoma N Engl J Med 364 2517ndash2526 (2011)

221 Stewart R et al The role of Fc gamma receptors in the activity of immunomodulatory antibodies for cancer J Immunother Cancer 2 29 (2014)

222 Gomez-Roca C A et al Phase I study of RG7155 a novel anti-CSF1R antibody in patients with advanced

R E V I E W S

NATURE REVIEWS | DRUG DISCOVERY VOLUME 14 | AUGUST 2015 | 583

copy 2015 Macmillan Publishers Limited All rights reserved

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 24: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

metastatic solid tumors J Clin Oncol 33 (Suppl abstr) 3005 (2015)

223 Simpson T R et al Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma J Exp Med 210 1695ndash1710 (2013)

224 Selby M J et al Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells Cancer Immunol Res 1 32ndash42 (2013)Shows that an ADCC-activating Fc region on the CTLA4-specific mAb is essential for antitumour activity in a mouse tumour model suggesting that the depletion of CTLA4-positive TReg cells within the tumour is a major mechanism

225 Mahoney K M amp Atkins M B Prognostic and predictive markers for the new immunotherapies Oncology (Williston Park) 28 (Suppl 3) 39ndash48 (2014)An overview of the differences between prognostic and predictive markers as well as the candidate predictive and pharmacodynamic markers in the field of immune checkpoint blockade focusing on PDL1 expression

226 Green M R et al Integrative analysis reveals selective 9p241 amplification increased PD-1 ligand expression and further induction via JAK2 in nodular sclerosing Hodgkin lymphoma and primary mediastinal large B-cell lymphoma Blood 116 3268ndash3277 (2010)This paper shows that genomic amplification of 9p241 encoding Janus kinase 2 PDL1 and PDL2 results in PDL1 and PDL2 overexpression in Hodgkin and primary mediastinal lymphomas

227 Ansell S M et al PD-1 blockade with nivolumab in relapsed or refractory Hodgkinrsquos lymphoma N Engl J Med 372 311ndash319 (2015)

228 Taube J M et al Association of PD-1 PD-1 ligands and other features of the tumor immune microenvironment with response to anti-PD-1 therapy Clin Cancer Res 20 5064ndash5074 (2014)

229 Paz-Ares L et al Phase III randomized trial (CheckMate 057) of nivolumab (NIVO) versus docetaxel (DOC) in advanced non-squamous cell (non-SQ) non-small cell lung cancer (NSCLC) J Clin Oncol 33 (Suppl) LBA109 (2015)

230 Brahmer J et al Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer N Engl J Med httpdxdoiorg101056NEJMoa1504627 (2015)

231 Schellens J et al CEA-targeted engineered IL2 clinical confirmation of tumor targeting and evidence of intra-tumoral immune activation J Clin Oncol 33 (Suppl) 3016 (2015)

232 Sznol M amp Chen L Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer-response Clin Cancer Res 19 5542 (2013)

233 Grosso J et al Programmed death-ligand 1 (PD-L1) expression in various tumor types J Immunother Cancer 1 (Suppl 1) P53 (2013)

234 Brown J A et al Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production J Immunol 170 1257ndash1266 (2003)Shows that enhanced T cell activation is a consequence of blockade of the interaction with PD1 not signal transduction by PDL1 or PDL2 The paper also presents data on PDL1 expression in a broad range of solid and haematological malignancies and normal tissues

235 Dong H et al Tumor-associated B7-H1 promotes T-cell apoptosis a potential mechanism of immune evasion Nat Med 8 793ndash800 (2002)The first paper to show PDL1 expression in situ on solid tumours including lung ovary colon and melanoma

236 Ohigashi Y et al Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2

expression in human esophageal cancer Clin Cancer Res 11 2947ndash2953 (2005)

237 Nomi T et al Clinical significance and therapeutic potential of the programmed death-1 ligandprogrammed death-1 pathway in human pancreatic cancer Clin Cancer Res 13 2151ndash2157 (2007)

238 Hamanishi J et al Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer Proc Natl Acad Sci USA 104 3360ndash3365 (2007)

239 Gao Q et al Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma Clin Cancer Res 15 971ndash979 (2009)

240 Latchman Y et al PD-L2 is a second ligand for PD-1 and inhibits T cell activation Nat Immunol 2 261ndash268 (2001)Identifies PDL2 as a second ligand for PD1 recruitment of SHP2 as a mechanism for PD1-mediated inhibition of T cell activation and first shows PDL1 expression on solid tumour cell lines

241 Shi M et al Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain Am J Surg Pathol 38 1715ndash1723 (2014)

242 Sun Y et al B7-H3 and B7-H4 expression in non-small-cell lung cancer Lung Cancer 53 143ndash151 (2006)

243 Wu C P et al Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis World J Gastroenterol 12 457ndash459 (2006)

244 Crispen P L et al Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma Clin Cancer Res 14 5150ndash5157 (2008)

245 Loos M et al Expression of the costimulatory molecule B7-H3 is associated with prolonged survival in human pancreatic cancer BMC Cancer 9 463 (2009)

246 Sun J et al Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma Cancer Immunol Immunother 59 1163ndash1171 (2010)

247 Zang X et al Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas Mod Pathol 23 1104ndash1112 (2010)

248 Roth T J et al B7-H3 ligand expression by prostate cancer a novel marker of prognosis and potential target for therapy Cancer Res 67 7893ndash7900 (2007)

249 Boorjian S A et al T-cell coregulatory molecule expression in urothelial cell carcinoma clinicopathologic correlations and association with survival Clin Cancer Res 14 4800ndash4808 (2008)

250 Tringler B et al B7-H4 is highly expressed in ductal and lobular breast cancer Clin Cancer Res 11 1842ndash1848 (2005)Although the receptor for B7-H4 is unknown this inhibitory B7 family member is highly expressed in breast cancer suggesting a mechanism of immune evasion and encouraging drug development

251 Krambeck A E et al B7-H4 expression in renal cell carcinoma and tumor vasculature associations with cancer progression and survival Proc Natl Acad Sci USA 103 10391ndash10396 (2006)

252 Kryczek I et al B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J Exp Med 203 871ndash881 (2006)

253 Simon I et al B7-H4 is a novel membrane-bound protein and a candidate serum and tissue biomarker for ovarian cancer Cancer Res 66 1570ndash1575 (2006)

254 Tringler B et al B7-H4 overexpression in ovarian tumors Gynecol Oncol 100 44ndash52 (2006)

255 Chen L J et al B7-H4 expression associates with cancer progression and predicts patientrsquos survival in

human esophageal squamous cell carcinoma Cancer Immunol Immunother 60 1047ndash1055 (2011)

256 Arigami T et al Clinical significance of the B7-H4 coregulatory molecule as a novel prognostic marker in gastric cancer World J Surg 35 2051ndash2057 (2011)

257 Jiang J et al Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer Cancer Immunol Immunother 59 1707ndash1714 (2010)

258 Wang Y et al α 1 antichymotrypsin is aberrantly expressed during melanoma progression and predicts poor survival for patients with metastatic melanoma Pigment Cell Melanoma Res 23 575ndash578 (2010)

259 Awadallah N S et al Detection of B7-H4 and p53 in pancreatic cancer potential role as a cytological diagnostic adjunct Pancreas 36 200ndash206 (2008)

260 Quandt D Fiedler E Boettcher D Marsch W amp Seliger B B7-H4 expression in human melanoma its association with patientsrsquo survival and antitumor immune response Clin Cancer Res 17 3100ndash3111 (2011)

261 Schulkens I A et al Galectin expression profiling identifies galectin-1 and galectin-9δ5 as prognostic factors in stage III non-small cell lung cancer PLoS ONE 9 e107988 (2014)

262 Lahm H et al Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures J Cancer Res Clin Oncol 127 375ndash386 (2001)

263 Lotan R et al Lactose-binding lectin expression in human colorectal carcinomas Relation to tumor progression Carbohydr Res 213 47ndash57 (1991)

264 Miyazaki J et al Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes Oncol Rep 9 1307ndash1312 (2002)

265 Pallesen G amp Hamilton-Dutoit S J Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma Am J Pathol 133 446ndash450 (1988)

266 Jilaveanu L B et al CD70 expression patterns in renal cell carcinoma Hum Pathol 43 1394ndash1399 (2012)

267 Yang Z Z Novak A J Ziesmer S C Witzig T E amp Ansell S M CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells Blood 110 2537ndash2544 (2007)

268 Schreiner B et al Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo Glia 44 296ndash301 (2003)

269 Martin-Orozco N et al Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells Cancer Res 70 9581ndash9590 (2010)

270 Sloan K E et al CD155PVR plays a key role in cell motility during tumor cell invasion and migration BMC Cancer 4 73 (2004)

AcknowledgementsGJF acknowledges research support from grants P50CA101942 U54CA163125 P01AI054456 and R01AI089955 KMM acknowledges research support from the Claudia Adams Barr Program for Innovative Cancer Research an American Association for Cancer Research Basic Cancer Research Fellowship (14-40-01-MAHO) and an American Society of Clinical Oncology Young Investigator Award supported by the Kidney Cancer Association

Competing interests statementThe authors declare competing interests see Web version for details

FURTHER INFORMATIONClinicalTrialsgov httpwwwclinicaltrialsgovJounce Therapeutics httpjouncetxcomPEGS Boston httpwwwpegsummitcom Immunotherapy-Targets

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

R E V I E W S

584 | AUGUST 2015 | VOLUME 14 wwwnaturecomreviewsdrugdisc

copy 2015 Macmillan Publishers Limited All rights reserved

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion
Page 25: CANCER IMMUNOTHERAPY Combination cancer immunotherapy …€¦ · a candidate for immunotherapy, is a landmark advance in cancer immunotherapy. Additionally, predictive bio-markers

Author biographiesKathleen M Mahoney is an instructor in the Department of Medicine at Harvard Medical School Boston Massachusetts USA She is a practicing oncologist specializing in genitourinary cancer at the Beth Israel Deaconess Medical Center Harvard Medical School and an active member of the Kidney Cancer Program at the DanandashFarber Cancer Institute Boston Massachusetts USA and Harvard Cancer Center Boston Massachusetts USA As a postdoctoral fellow at the DanandashFarber Cancer Institute in the laboratory of Gordon J Freeman her research interest lies in improving the understanding of the pro-grammed cell death 1 ligand 1 (PDL1) pathway in tumour biology investigating potential predictive markers for immune checkpoint blockade and exploring combination therapies that may overcome resistance to immune checkpoint blockade

Paul D Rennert is a biotechnology executive and strategic leader with extensive biologic and small-molecule drug discovery experience in inflammation autoimmunity fibrosis oncology and immuno-oncol-ogy He co-founded and is Chief Scientific Officer (CSO) of Videre Biotherapeutics Watertown Massachusetts USA an oncology drug development company He founded and is the managing partner at SugarCone Biotech Holliston Massachusetts USA a consulting firm that provides strategic counselling for the biopharma and investment communities His prior positions include CSO at X-Rx Waltham Massachusetts USA principal investigator in immunology and inflammation at Biogen Cambridge Massachusetts USA molecular biologist at Repligen Cambridge Massachusetts USA and research associate at the Howard Hughes Medical Institute Massachusetts General Hospital Boston Massachusetts USA

Gordon J Freeman is Professor of Medicine at Harvard Medical School Boston Massachusetts USA and has worked at the DanandashFarber Cancer Institute Boston Massachusetts USA since 1979 His work has identified the major pathways inhibiting and co-stimulating T cell activation He and three others received the Cancer Research Institutersquos 2014 William B Coley Award for Distinguished Research in Tumour Immunology for the following discoveries programmed cell death protein 1 (PD1) is a new immune checkpoint on T cells this receptor has two natural ligands or binding partners called pro-grammed cell death 1 ligand 1 (PDL1) and PDL2 one of these PDL1 is often found in human cancer and blocking either receptor or ligand with a monoclonal antibody could result in improved immune-related tumour destruction

Competing interests statementGJF has patents pending royalties on the programmed cell death protein 1 (PD1) pathway from Bristol-Myers Squibb Roche Merck EMD-Serono Boehringer-Ingelheim AstraZeneca and Novartis GJF has served on advisory boards for CoStim Novartis Roche and Bristol-Myers Squibb KMM and PDR declare no competing interests

Key pointsbull Tumours actively inhibit the antitumour immune responsebull The programmed cell death protein 1ndashprogrammed cell death 1

ligand 1 (PD1ndashPDL1) pathway has a natural role in regulating peripheral tolerance and restraining over-exuberant immune responses

bull PDL1 is a critical dominant immunoinhibitor in many tumour

types leading to immune evasion by the tumour PD1 pathway blockade unleashes a previously exhausted immune response that is focused on tumour neoantigens

bull Immunotherapies that increase the antitumour immune response such as interleukin-2 or blockade of cytotoxic T lymphocyte anti-gen 4 (CTLA4) PD1 or PDL1 can benefit a moderate number of patients with cancer with a durable clinical benefit in some

bull Many patients fail to develop tumour shrinkage when blocking only one immune checkpoint and increasing response rates is a major and achievable goal

bull Given the tolerability and efficacy of PD1 pathway blockade in clinical trials it is a good foundation for combination therapies seeking to increase response rates

bull Once PD1 or CTLA4 are blocked many other therapies can aug-ment their efficacy including blockade of other immunoinhibi-tory pathways stimulation by activating pathways such as tumour necrosis factor receptor superfamily members some chemothera-pies radiation epigenetic modifiers targeted therapies angiogen-esis blockade augmentation of natural killer cell activity chimeric antigen receptor T cell therapies and vaccines

bull Therapeutics may work by blocking immunoinhibitory targets or stimulating immunoactivating targets not only on lymphocytes but also on macrophage natural killer and stromal cells to overcome failure to respond to single checkpoint blockade

ToC blurb

000 Combination cancer immunotherapy and new immunomodulatory targetsKathleen M Mahoney Paul D Rennert and Gordon J FreemanAnticancer immunotherapy through checkpoint blockade enables the patient to mount active antitumour responses and can dramatically improve survival In this Review Mahoney Rennert and Freeman examine targets for next-generation immunomodulators and discuss how these may be integrated in rational combination therapies with existing and upcoming immune-targeted drugs

O N L I N E O N LY

copy 2015 Macmillan Publishers Limited All rights reserved

  • Abstract | Targeting immune checkpoints such as programmed cell death protein 1 (PD1) programmed cell death 1 ligand 1 (PDL1) and cytotoxic T lymphocyte antigen 4 (CTLA4) has achieved noteworthy benefit in multiple cancers by blocking immunoinhibitory si
  • Box 1 | Autoimmunity and the anticancer response by-product or bystander
  • Figure 1 | T cell activation is a multiple-signal processenspThe T cell receptor (TCR) starts the signalling cascade upon its interaction with peptide antigen in the context of the major histocompatibility complex (MHC) (signal 1) but optimal activation of
  • Figure 2 | Tumours can express co-inhibitory and co-stimulatory ligandsenspIndividual tumours can express a selected subset of co‑inhibitory ligands which aid in evading the antitumour immune response HHLA2 HERV-H LTR-associating protein 2 ICOSL induci
  • Checkpoint inhibition why combinations
  • Checkpoint blockade as a backbone
  • Table 1 | Immunological targets currently in clinical or preclinical development
  • Table 1 (cont) | Immunological targets currently in clinical or preclinical development
  • Box 2 | Lessons learnt from CTLA4 inhibitors
  • New targets the TNFR superfamily
  • Box 3 | Considerations in the design of immunomodulatory antibodies
  • Box 4 | Biomarkers for checkpoint inhibitor responses
  • Figure 3 | Immunoregulatory receptors expressed on the cell surface of regulatory T cellsenspRegulatory T cells express several immunomodulatory receptors that can be targeted by immunotherapies For example therapeutic antibodies such as the cytotoxic T
  • Novel IgSF proteins
  • B7 and CD28‑related proteins
  • TIGIT and PVR family members
  • Figure 4 | Activating and inhibitory receptors on natural killer cellsenspNatural killer (NK) cells express both activating and inhibitory receptors that can interact with ligands on antigen presenting cells or tumour cells to modulate NK cell function For
  • Immunomodulatory targets on natural killer cells
  • Targeting the tumour microenvironment
  • Figure 5 | Immunosuppressive factors in the tumour microenvironmentenspIn addition to cellndashcell interactions that can inhibit effector lymphocytes soluble factors can suppress the local immune response which creates a hostile environment for infiltrating
  • Targeting cell discrimination mechanisms
  • Conclusion