targetingcd73enhancestheantitumoractivityofanti-pd-1 and ... · inhibitors hold great potential as...

Cancer Therapy: Preclinical

TargetingCD73Enhances theAntitumorActivity of Anti-PD-1and Anti-CTLA-4 mAbs

Bertrand Allard1, Sandra Pommey1, Mark J. Smyth2,3,4,5, and John Stagg1

AbstractPurpose: Monoclonal antibodies (mAb) that block programmed death (PD)-1 or cytotoxic T

lymphocyte antigen (CTLA-4) receptors have been associated with durable clinical responses against

a variety of cancer types and hold great potential as novel cancer therapeutics. Recent evidence suggest

that targeted blockade of multiple immunosuppressive pathways can induce synergistic antitumor

responses.

Experimental Design: In this study, we investigated whether targeted blockade of CD73, an ectonu-

cleotidase that catabolizes the hydrolysis of extracellular adenosine monophosphate (AMP) to adenosine,

can enhance the antitumor activity of anti-CTLA-4 and anti-PD-1mAbs against transplanted and chemically

induced mouse tumors.

Results: Anti-CD73 mAb significantly enhanced the activity of both anti-CTLA-4 and anti-PD-1 mAbs

against MC38-OVA (colon) and RM-1 (prostate) subcutaneous tumors, and established metastatic 4T1.2

breast cancer. Anti-CD73 mAb also significantly enhanced the activity of anti-PD-1 mAb against 3-

methylcholanthrene (MCA)-induced fibrosarcomas. Gene-targeted mice revealed that single-agent thera-

pies and combinatorial treatments were dependent on host IFN-g and CD8þ T cells, but independent of

perforin. Interestingly, anti-CD73 mAb preferentially synergized with anti-PD-1 mAb. We investigated the

effect of extracellular adenosine on tumor-infiltrating T cells and showed that activation of A2A adenosine

receptor enhances PD-1 expression, but not CTLA-4 expression, on tumor-specific CD8þ T cells and CD4þFoxp3þ T regulatory cells.

Conclusions: Taken together, our study revealed that targeted blockade of CD73 can enhance the

therapeutic activity of anti-PD-1 and anti-CTLA-4 mAbs and may thus potentiate therapeutic strategies

targeting immune checkpoint inhibitors in general. Clin Cancer Res; 19(20); 5626–35. �2013 AACR.

IntroductionTumor-infiltrating lymphocytes (TIL) control the clinical

progression of various types of cancers (1–4). Nevertheless,most tumors persist despite being infiltrated with tumor-specific CD8þ T cells. This is in part due to the dysfunctional

nature of TILs and the presence of immunosuppressivefactors and regulatory cells in the tumormicroenvironment(5, 6). One of the major causes of TIL dysfunction is aprocess known as T-cell exhaustion, which results in theexpression of inhibitory receptors (7–9). Monoclonal anti-bodies (mAb) able to block these inhibitory receptors—alsocalled immune-checkpoint inhibitors—have been associat-ed with objective clinical responses against various types ofcancer and hold great potential as novel cancer therapeutics(10–12).

The first immune-checkpoint inhibitor approved for can-cer treatment is the anti-CTLA-4 mAb ipilimumab (Yervoy;ref. 13). CTLA-4 belongs to the immunoglobulin superfam-ily of receptors, which also includes PD-1, TIM-3 (T-cellimmunoglobulin and mucin domain-containing protein3), BTLA (B and T lymphocyte attenuator), and VISTA (V-domain immunoglobulin suppressor of T-cell activation;ref. 14). Ipilimumab is approved for treatment of unresect-able or metastatic melanoma, either as initial therapy orafter relapse. Anti-CTLA-4 mAb therapy enhances the anti-tumor function of CD8þ T cells, increases the ratio of CD8þ

T cells to Foxp3þ T regulatory cells (Tregs) and inhibitsthe suppressive function of Tregs (15). Another promising

Authors' Affiliations: 1Centre de Recherche du Centre Hospitalier del'Universit�e de Montr�eal, Facult�e de Pharmacie et Institut du Cancer deMontr�eal, Montr�eal, Qu�ebec, Canada; 2Cancer Immunology Program,Trescowthick Laboratories, Peter MacCallum Cancer Centre, St. AndrewsPlace, East Melbourne; 3Sir Peter MacCallum Department of Oncology,University of Melbourne, Parkville, Victoria; 4Immunology in Cancer andInfection Laboratory, Queensland Institute of Medical Research; and5School of Medicine, University of Queensland, Herston, Queensland,Australia

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

M.J. Smyth and J. Stagg are co-senior authors of this article.

Corresponding Author: John Stagg, Centre de recherche du CentreHospitalier de l'Universit�e deMontr�eal, Montr�eal, Qu�ebec, Canada. Phone:514-890-8000, ext: 25170; Fax: 514-412-7661; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-0545

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(20) October 15, 20135626

on January 20, 2020. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

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immune checkpoint that can be targeted for cancer therapyis the PD-1/PD-L1 axis (16, 17). PD-1 is expressed byactivated T cells, B cells, monocytes, and NKT cells. It hastwo ligands, PD-L1, and PD-L2, with distinct expressionprofiles. Notably, PD-L1 expression on tumor cells has beenassociated with poor prognosis in patients with cancer (18,19). Targeted blockade of PD-1 or PD-L1 enhances adaptiveantitumor immunity bypreventing T-cell exhaustion andbypromoting T-cell homing to tumors (20, 21). In two recentphase I clinical trials, targeted blockade of PD-1/PD-L1 wasassociatedwith objective clinical responses in 6% to 28%ofpatients with cancer (16, 17).While somepatients respond to anti-CTLA-4 or anti-PD-1

mAb therapy, the majority of patients show modest or noclinical benefits. One potential explanation is that tumorsuse additional and nonoverlapping immunosuppressivemechanisms to facilitate immune escape (5). Targetedblockade of multiple immunosuppressive pathways mightthus be required to induce clinically relevant antitumorresponses. This is supported by several recent studies show-ing that targeted inhibition ofmultiple immunosuppressivepathways can induce synergistic anticancer activity in mice(21–23).One of the most recently identified immunosuppressive

pathways involved in tumor immune escape is the produc-tion of extracellular adenosine by CD73 (ecto-50-nucleo-tidase; refs. 24–26). CD73 is expressed on lymphocytes,endothelial, and epithelial cells, where it participates inendothelial cell barrier function, protection from ischemia,and regulation of immune responses. Importantly, CD73has been found to be overexpressed in several types ofhuman cancers, and high CD73 expression has been asso-ciated with a poor prognosis (27). We and others haveshown that CD73 expressed on tumor cells as well as onhost hematopoietic and nonhematopoietic cells promotestumor immune escape and metastasis in mice (28–31). Wehave also established the proof-of-concept that targeted

blockade of CD73 can induce potent antitumor immuneresponses (32) and synergize with chemotherapeutic drugsknown to promote immunogenic responses (33). The inhi-bition of CD39, an ectoenzyme operating upstream ofCD73, was also shown to have therapeutic effects and tosynergize with chemotherapy in preclinical cancer models(34).

Herein, we investigated whether targeted blockade ofCD73 can enhance the therapeutic activity of the immunecheckpoint inhibitors anti-CTLA-4 and anti-PD-1 mAbs.Our study reveals that anti-CD73 mAb enhances thetherapeutic activity of both anti-CTLA-4 and anti-PD-1mAbs. Interestingly, the greatest therapeutic effect wasobserved when anti-CD73 mAb was combined with anti-PD-1 mAb. Analysis of tumor-infiltrating T cells revealedthat CD73-derived adenosine enhances PD-1, but notCTLA-4 expression on TILs via activation of A2A adeno-sine receptor. Taken together, our study suggests thattargeted blockade of CD73 can synergize with immunecheckpoint inhibitors, especially those targeting thePD-1/PD-L1 axis.

Materials and MethodsCell lines and animals

MC38-OVA (CD73low), RM-1 (CD73low), and 4T1.2(CD73high) tumor cell lines have been previously described(28, 29, 32). Wild-type C57Bl/6 or BALB/c mice werepurchased fromCharles River andmaintained at the Centrede Recherche du Centre Hospitalier de l’Universit�e deMontr�eal, or bred and maintained at the Peter MacCallumCancer Centre. IFN-g–deficient C57Bl/6mice and perforin-deficient C57Bl/6 mice were bred and maintained at thePeter MacCallum Cancer Centre. CD73-deficient mice werekindly provided by Dr. Linda H. Thompson (OklahomaMedical Research Foundation, Oklahoma City, OK) andwere bred and maintained at the Centre de Recherche duCentre Hospitalier de l’Universit�e de Montr�eal.

Antibodies and chemicalsPurified anti-mouse CD73 mAb (clone TY/23), purified

anti-mouse PD-1 mAb (clone RMP1-14), purified anti-mouse CTLA-4 mAb (clone 9H10 and UC10-4F10), anti-mouse CD4 (clone GK1.5), anti-mouse CD8b (clone53.5.8), and purified control Ig (MAC4) were purchasedfrom BioXCell. V450-conjugated anti-mouse CD8a, PE-CF594–conjugated anti-mouse PD-1, Alexa488-conjugatedanti-mouse CTLA-4, Alexa647-conjugated anti-mouseTCRb, Alexa700-conjugated anti-mouse CD4, PE-conjugat-ed anti-mouse Foxp3, PE- or Alexa647-conjugated anti-mouse CD73 (clone TY/11.8) were purchased from BDBioscience. PE-conjugated MHC class I/SIINFEKL tetramerwas obtained from Universit�e de Montr�eal. 50-(N-ethylcar-boxamido) adenosine (NECA) and SCH58261 were pur-chased from Sigma.

In vivo treatmentsMC38-OVA tumor cells (106 cells) or RM-1 tumor cells

(5� 105 cells) were injected subcutaneously into syngeneic

Translational RelevanceSome patients with cancer show durable clinical

responses to anti-CTLA-4 or anti-PD-1monoclonal anti-bodies (mAb). For this reason, immune-checkpointinhibitors hold great potential as cancer therapeutics.Nevertheless, clinical benefits from immune-checkpointinhibition are still modest. One potential explanation isthat tumors use nonoverlapping immunosuppressivemechanisms to facilitate immune escape. One of thesemechanisms consists at the accumulation of extracellu-lar adenosine. In this study, we show that targetedblockade of CD73, the ectonucleotidase that catabolizesthe generation of extracellular adenosine, significantlyenhances the therapeutic activity of anti-PD-1 and anti-CTLA-4 mAbs. Our study strongly thus suggests thattargeted blockade of CD73 can improve the antitumoractivity of immune checkpoint inhibitors.

Anti-CD73 Synergizes with Anti-PD-1 and Anti-CTLA-4 mAbs

www.aacrjournals.org Clin Cancer Res; 19(20) October 15, 2013 5627




C57Bl/6mice.Where indicated,micewere treatedwith anti-CD73 mAb (TY/23; 100 mg, i.p.), anti-PD-1 mAb (RMP1-14; 100 mg, i.p.), anti-CTLA-4 mAb (9H10 or UC10-4F10;100 mg, i.p), or control Ig (MAC4; 100 mg i.p). To assessthe role of T cells, mice were also injected weekly intra-peritoneally with anti-CD4 mAb (100 mg clone GK1.5)and/or anti-CD8b mAb (100 mg clone 53.5.8) or controlIg (100 mg clone MAC4). For survival experiments, 5 �104 4T1.2 tumor cells were inoculated into the fourthmammary fat pad of BALB/c mice. On day 25, mice wereanesthetized, the primary tumor was surgically removed,the wound was closed with surgical clips, and treatmentscommenced 3 days later. To assess PD-1 and CTLA-4expression levels in TILs, MC38-OVA tumor cells (106

cells) were injected subcutaneously into syngeneic wild-type or CD73-deficient C57Bl/6 mice, treated at day 12and 15 with SCH58261 (1 mg/kg) and/or NECA (0.05mg/kg i.p. þ 0.05 mg/kg, s.c.) and tumors isolated at day18 for flow cytometry analysis.

Flow cytometry of tumor-infiltrating lymphocytesMC38-OVA tumors were excised, minced with scissors,

and incubated 1 hour at 37�C for in PBS containingcollagenase type 4 (Worthington Biochemical) andDNase I (Roche). Tumor cell suspensions were passedthrough a 70-mm cell strainer, washed twice in PBS, andresuspended in PBS 2% serum. Anti-CD16/32 mAb(clone 2.4G2) was used to block Fc receptors. To assessovalbumin-specific CD8þ T cells, TCRbþCD8aþ cellswere pregated and percentages of tetramer-reactive cellsevaluated. Flow cytometry was conducted on a Fortessa(BD Bioscience) and analyzed using the software programFlowJo.

Quantitative real-time PCRTumor tissues were disrupted by a rotor-stator homog-

enizer followed by RNA isolation using RNAeasy MiniKit (Qiagen) according to the manufacturer’s instruc-tions. cDNA reverse transcription was carried out using

Figure 1. Anti-CD73mAb enhances antitumor activity of anti-PD-1 and anti-CTLA-4mAbs.Wild-type (A and D), IFN-g–deficient (B and E) or perforin-deficient(C and F) C57Bl/6 mice were injected subcutaneously with MC38-OVA tumor cells (106 cells) and treated on day 12, 16, and 20 with intraperitoneal(i.p.) injections of anti-CD73 mAb (100 mg clone TY/23), anti-PD-1 mAb (100 mg clone RMP1-14), anti-CTLA-4 mAb (100 mg clone UC10-4F10),anti-CD73/anti-PD-1 mAbs (100 mg i.p. each; A-C), anti-CD73/anti-CTLA-4 mAbs (100 mg i.p each; D–F) or control Ig (200 mg, i.p.). Means � SEs of5mice per group are shown (�,P < 0.05 byMann–Whitney between combination and either single-agent therapy). A representative of 2 experiments is shown.

Allard et al.

Clin Cancer Res; 19(20) October 15, 2013 Clinical Cancer Research5628




Superscript VILO cDNA Synthesis Kit (Invitrogen). Real-time PCR was carried out using a Step One Plus Thermalcycler using oligonucleotides and TaqMan probe mixspecific (gene expression assays) for mouse IFN-g(Mm01168134_m1), Tbx21 (Mm00450960_m1), andGADD45A (Mm00432802_m1; all from Applied Biosys-tems). Samples were normalized relative to GADD45Atranscript expression levels.

MCA-induced fibrosarcomasGroups of 15 male C57Bl/6 mice were inoculated sub-

cutaneously in the hind flank with 400 mg of 3-methylcho-lanthrene (MCA; Sigma-Aldrich) in 0.1 ml of corn oil.Once the tumors were established (0.24–0.39 cm2) micewere treated with intraperitoneal injections of cIg (100mg), anti-CD73 mAb (100 mg), anti-PD-1 mAb (100 mg),or combination anti-CD73/PD-1 mAbs (100 mg each)twice weekly for 6 weeks. Development of fibrosarcomaswas monitored weekly over the course of 300 days.Measurements were made with a caliper and tumor sizeswere calculated using the product of two perpendiculardiameters (mm2).

ResultsCD73 blockade enhances anti-PD-1/CTLA-4 mAbtherapy via IFN-g and CD8þ T cells

We investigated whether targeted blockade of CD73could enhance the therapeutic activity of anti-PD-1 andanti-CTLA-4 mAbs. In a first set of experiments, MC38mouse colon cancer cells expressing ovalbumin (MC38-OVA) were injected subcutaneously to syngeneic mice andtreated on day 12, 16, and 20 with anti-CD73 and/or anti-PD-1 mAbs. Monotherapy with anti-CD73 or anti-PD-1mAb significantly delayed tumor growth, consistent withprevious studies (28, 20). Strikingly, the combined admin-istration of anti-CD73 and anti-PD-1 mAbs induced com-plete tumor rejection in all treated mice (Fig. 1A). Weinvestigated the importance of IFN-g andperforin responsesin this anti-tumor effect using gene-targeted mice andobserved that the antitumor activity of anti-PD-1 andanti-CD73 mAbs, administered as single agents or in com-bination, was dependent on IFN-g (Fig. 1B) and indepen-dent of perforin (Fig. 1C).

We next investigated the effect of combining anti-CD73mAb with anti-CTLA-4 mAb. As shown in Fig. 1D, anti-

Figure 2. Anti-CD73 mAb enhancesTh1 responses generated by anti-PD-1 and anti-CTLA-4 mAbs. A,wild-type C57Bl/6 mice wereinjected subcutaneously withMC38-OVA tumor cells (106 cells)and treated on day 12 and 15 withintraperitoneal (i.p.) injections ofanti-CD73 mAb (100 mg cloneTY/23), anti-PD-1 mAb (100 mgcloneRMP1-14), anti-CTLA-4mAb(100 mg clone 9H10), anti-CD73/anti-PD-1 mAbs (100 mg each),anti-CD73/anti-CTLA-4 mAbs(100mgeach), or control Ig (200mg).On day 18, tumor single-cellsuspensionswere analyzedby flowcytometry for CD8 expression andtetramer reactivity (Tet). Means �SEs of 5 mice per group are shown(�, P < 0.05 by Mann–Whitney test).B, real-time PCR analysis ofIFN-g and T-bet (Tbx21) mRNAexpression in tumors aftertreatment. Means � SEs of 4 miceper group are shown (�, P < 0.05 byMann–Whitney test). C and D,percentages (C) and meanfluorescent intensity (MFI; D) ofCD73 expression in gated TILpopulations from MC38-OVAtumor-bearing mice treatedintraperitoneally with anti-CD73mAb or control Ig. Different anti-CD73 mAbs were used forblockade (clone TY/23) and flowcytometry (TY/11.8). Means � SEsof 5 mice per group are shown(��, P < 0.001 by Mann–Whitneytest).






CD73 mAb significantly enhanced the therapeutic activityof anti-CTLA-4 mAb (Fig. 1D). However, it did not inducecomplete tumor regression. Similar to anti-PD-1 mAb, theantitumor activity of anti-CTLA-4 and anti-CD73 mAbs,administered as single agents or in combination, wasdependent on IFN-g (Fig. 1E) and independent of perforin(Fig 1F). Similar to what we observed in MC38-OVAtumors, anti-CD73 mAb significantly enhanced the antitu-mor activity of anti-PD-1 and anti-CTLA-4 mAbs againstsubcutaneous RM-1 prostate tumors (Supplementary. Fig.S1A and S1B). We further investigated the role of CD8þ Tcells and CD4þ T cells in the activity of anti-CD73 mAb assingle agent or in combination with anti-CTLA-4 or anti-PD-1mAb.Our data showed that treatmentwith anti-CD73mAb as a single agent or in combination with anti-CTLA-4or anti-PD-1 mAb is dependent on CD8þ T cells, indepen-dently of CD4þ T cells (Supplementary Fig. S2).

Anti-CD73 mAb potentiates Th1 responses anddownregulates CD73 expression on TILs

We next investigated the effect of the combinatorialtreatments on tumor-infiltrating antigen-specific CD8þ Tcells. Consistent with previous studies (21, 32), mono-therapy with anti-CD73, anti-CTLA-4 or anti-PD-1 mAbincreased tumor infiltration with antigen-specific CD8þ Tcells compared with control-treated mice (Fig. 2A). Whenanti-CD73 mAb was combined with anti-PD-1 or anti-CTLA-4 mAb, tumor-infiltrating antigen-specific CD8þ

T cells further increased compared with single agenttreatments (Fig. 2A). Notably, this increased accumula-tion of antigen-specific CD8þ T cells following combina-torial treatments was associated with increased expressionof the Th1 immune genes Tbx21 (also called T-bet) andits target IFN-g (Fig. 2B). We had previously shown thatanti-CD73 mAb can downregulate CD73 expression ontumor cells (32). We now investigated whether anti-CD73mAb therapy downregulated CD73 expression on TILs.As shown in Fig. 2C and D (and Supplementary Fig. S3),anti-CD73 mAb therapy significantly downregulatedCD73 expression levels on CD8þ and CD4þ TILs, irre-spective of tumor specificity or Foxp3 expression.

CD73 blockade prolongs survival of mice withmetastatic cancer when combined with anti-PD-1/CTLA-4 mAbs

We next assessed the therapeutic activity of combininganti-CD73 mAb with anti-PD-1 or anti-CTLA-4 mAb in amouse model of established metastatic cancer. Syngeneicmicewere injected subcutaneouslywith 4T1.2mouse breastcancer cells (which have the potential to spontaneouslymetastasize), primary tumors were surgically removed atday 25 and immunotherapy was initiated at day 28. Asshown in Fig. 3, tumor-bearing mice undergoing surgeryalone, with subsequent control treatment, died of metastat-ic disease by day 41 (median survival of 36 days). Post-surgerymonotherapywith anti-CD73mAb, anti-PD-1mAbor anti-CTLA-4 mAb each significantly prolonged survivalcompared with control treatment (median survival of 46.5,

55, and 49 days, respectively). When anti-CD73 mAb wascombined with anti-CTLA-4 mAb, the median survival wasincreased to 63 days. When anti-CD73 mAb was combinedwith anti-PD-1 mAb, the median survival was increased to70 days (Fig. 3; significance of differences in survivalbetween groups estimated by Log-rank Mantel–Cox test,P < 0.0001).

Combined treatment with anti-CD73/anti-PD-1 mAbsinduces tumor regression in a chemically inducedsarcoma model

We further assessed whether targeted blockade ofCD73 could enhance the therapeutic activity of immunecheckpoint inhibition with anti-PD-1 mAb in a more strin-gent model of carcinogenesis. We used the MCA-inducedfibrosarcomamodel, where host immunity has been shownto suppress tumor initiation and progression, and whereCD73 has been shown to promote tumor immune escape(29). C57Bl/6 mice were inoculated with 400 mg MCAsubcutaneously and were treated with anti-CD73 mAb,anti-PD-1 mAb, or combination anti-CD73/anti-PD-1mAbs for 6 weeks. As shown in Fig. 4, while single agenttreatments had modest antitumor effects, combining anti-CD73mAbwith anti-PD-1mAb significantly delayedMCA-induced tumor progression. Comparing individual tumorgrowth rates (in mm2/day) also revealed greater antitumoractivity with the combination treatment (P < 0.0001 byANOVA or Kruskal–Wallis test; Supplementary Fig. S4).

Figure3. Therapeutic activity of combining anti-CD73mAbwith anti-PD-1or anti-CTLA-4 mAb for treatment of established metastatic breastcancer. Fifty thousand 4T1.2 tumor cells were inoculated into themammary fat pad of wild-type BALB/c mice, primary tumors weresurgically removed on day 25 and mice were treated with 100 mg anti-CD73 mAb (TY/23), anti-PD-1 mAb (RMP1-14), and/or anti-CTLA-4 mAb(UC10-4F10) on day 28, 32, 36, and 40. Significance of differences insurvival between all groups was estimated by log-rank (Mantel–Cox) test(P < 0.0001). Comparing anti-CD73 versus anti-PD-1/anti-CD73 or anti-CD73/anti-CTLA-4; P ¼ 0.0002 by log-rank and P ¼ 0.002 by Gehan–Breslow–Wilcoxon (GBW) test. Comparing anti-CTLA-4 versus anti-CD73/anti-CTLA-4, P ¼ 0.0027 by log-rank and P ¼ 0.0043 by GBW.Comparing anti-PD-1 versus anti-PD1/anti-CD73, P ¼ 0.0027 by log-rank and P ¼ 0.0043 by GBW. Comparing anti-PD-1/anti-CD73 versusanti-CD73/anti-CTLA-4,P¼ 0.0018by log-rank andP¼ 0.0039 byGBW.

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Notably, 2 of 15 mice treated with the combination anti-CD73/anti-PD-1 mAbs showed complete tumor regressionand 6 of 15 mice showed a partial response.

Activation of A2A adenosine receptor upregulates PD-1expression on TILsBecause extracellular adenosine often accumulates in

solid tumors and that TILs often express PD-1 and/orCTLA-4, we investigated whether CD73-derived adenosinecould regulate PD-1 and/or CTLA-4 expression in TILs. Forthis purpose, we treated tumor-bearing mice with the pan-adenosine receptor agonist NECA and analyzed cell surfaceexpression of PD-1 and CTLA-4 on TILs. PD-1 and CTLA-4expression was also assessed on TILs of CD73-deficientmice. As shown in Fig. 5, administration of NECA totumor-bearingmice significantly increased PD-1 expressionlevels on antigen-specific CD8þ TILs and CD4þFoxp3þ

TILs, but had no effect on CTLA-4 expression. Conversely,in tumor-bearing CD73-deficient mice, CD8þ TILs showeddecreased expression of PD-1, but not CTLA-4 (Fig. 5B),whereas CD4þFoxp3þ TILs showed unaltered PD-1 andCTLA-4 expression (Fig. 5C). Because high affinity A2Aadenosine receptor is upregulated on activated T cells, weinvestigated whether A2A adenosine receptor signaling wasinvolved in upregulating PD-1 expression on TILs. Asshown in Fig. 5B, NECA-mediated upregulation of PD-1

on TILs was inhibited when mice were cotreated with anA2A adenosine receptor antagonist (SCH58261). We fur-ther investigated the increase in PD-1 expression levels on Tcells induced by A2A adenosine receptor signaling in vitro.As shown in Fig. 5D, adenosine receptor activation withNECA following in vitro T-cell activation significantlyincreased PD-1, but not CTLA-4, expression levels on bothCD4þ and CD8þ T cells, and this was completely inhibitedby the A2A adenosine receptor antagonist SCH58261. Tak-en together, our data strongly suggest that CD73-derivedadenosine enhances PD-1 expression on activated T cells viaA2A adenosine receptor signaling.

Our data suggested that anti-CD73 mAb may decreasePD-1 levels in TILs. To test this hypothesis, we analyzed PD-1 and CTLA-4 expression levels in MC38-OVA TILs aftertreatment with anti-CD73mAb. Treatment with anti-CD73mAb did not alter PD-1 or CTLA-4 levels in TILs (Supple-mentary Fig. S5). Thus, while extracellular adenosine pos-itively regulates PD-1 levels on TILs, blocking CD73 withmAb clone TY/23 is insufficient to affect this process,suggesting a nonredundant mechanism of synergy betweenTY/23 and anti-PD-1 mAb.

DiscussionIn this study, we report that targeted blockade of CD73

can enhance the therapeutic activity of the immune-

Figure 4. Combined anti-CD73/anti-PD-1 mAb therapy inhibitscarcinogen-inducedtumorigenesis. C57Bl/6 micewere inoculated subcutaneously inthe hind flank with 400 mg of 3-methylcholanthrene (MCA). Oncetumors were established (0.24–0.39 cm2), mice were treated with(A) intraperitoneal (i.p.) injections ofcontrol Ig (100 mg), (B) anti-CD73mAb (100 mg TY/23), (C) anti-PD-1mAb (100 mg RMP1-14), or (D)combination anti-CD73/PD-1mAbs (100 mg each) twice weeklyfor 6 weeks. Development offibrosarcomas was monitoredweekly over the course of 300days. Tumor sizes of individualmice are represented.






Figure 5. CD73 and A2A adenosinereceptor signaling enhances PD-1expression on tumor-infiltrating Tcells. Wild-type or CD73-deficientC57Bl/6 mice were injectedsubcutaneously with MC38-OVAtumor cells (106cells) and treated atday 12 and 15 with the pan-adenosine receptor agonist NECA(0.05 mg/kg i.p. þ 0.05 mg/kg s.c.)and/or the A2A adenosine receptorantagonist SCH58261 (1 mg/kg).At day 18, tumor cell suspensionswere analyzed. A, FACS dot plotsof tumor-infiltrating CD8þ T cells(left) and histograms (right) of PD-1expression levels on gated CD8þ

Tetþ T cells. B and C, meanfluorescent intensity (MFI) of PD-1(left) and CTLA-4 (right) expressionon ovalbumin-specific CD8þ Tcells (B) and CD4þFoxp3þ T cells(C). Means � SEs of 5 mice pergroup are shown (�, P < 0.05 byMann–Whitney test). D, splenicC57Bl/6 T cells were activated invitro and treated 48 hours later withNECA � SCH58261. PD-1 andCTLA-4 expression levels (MFI)were measured by flow cytometryon CD4þ and CD8þ T cells at 24,48, and 72 hours postactivation.

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checkpoint inhibitors anti-PD-1 and anti-CTLA-4 mAbs.Blocking immune checkpointswithmonoclonal antibodiesis associated with objective clinical responses in a variety ofcancer types (10, 13, 16, 17). The anti-CTLA-4 mAb ipili-mumab is approved for treatment of metastatic melanomaand is being evaluated for other cancer indications (clin-icaltrial.gov identifiers: NCT01693783, NCT01688492,NCT01611558,NCT01285609).While ipilimumab is asso-ciated with clinical responses, it is limited by the generationof autoimmune toxicities due to on-target effects. Accord-ingly, up to 25% of patients treated with ipilimumabdevelop serious grade 3 to 4 adverse events (35). TargetingPD-1 or its ligand PD-L1 is as another promising cancerimmunotherapy. Remarkably, in phase I clinial trials (16,17), anti-PD-1/anti-PD-L1 mAbs were associated withobjective clinical responses in a variety of cancer types,including poorly immunogenic cancers such as non–smallcell lung cancer. Anti-PD-1/anti-PD-L1mAb therapy causeddrug-related grade 3 or 4 adverse events in 14% and 9% ofpatients, respectively (16, 17). Thus, a major challenge inthe use of anti-CTLA-4 and anti-PD-1/PD-L1 mAbs is todefine favorable clinical settings that strike an optimalbalance between tumor immunity and autoimmunity.One potential avenue is to target nonoverlapping immu-

nosuppressive mechanisms that facilitate immune escape.Theoretically, targeting immunosuppressive pathwaysspecifically expressed in the tumor microenvironmentwill favor the activation of antitumor over autoimmuneresponses. Targeted blockade of multiple immunosuppres-sive pathways might thus be required to induce clinicallyrelevant antitumor responses. Indeed, several studies haveshowed the synergistic antitumor activity of combinatorialimmunotherapies (22, 23, 36, 37). Curran and colleagues(21) showed that combined blockade of CTLA-4 and PD-1induces synergistic antitumor activity in mice. Concurrentblockade of CTLA-4 and PD-1 has also shown encouragingresults in a phase I clinical trial (37). Studies have also testedthe potential benefit of combining anti-PD-1 mAb withtumor vaccines (38, 39) and low-dose Treg-depleting cyclo-phosphamide (40). In the latter setting, anti-PD-1mAbwasfound to prolong the inhibition of Tregs by low-dosecyclophosphamide. Anti-PD-1 mAb therapy has also beenshown to act synergistically with adoptive T-cell therapy(41, 42).Our data demonstrated that both anti-PD-1 and anti-

CTLA-4 mAbs can significantly benefit from targeted block-ade of CD73. In the subcutaneous MC38-OVA tumormodel, we observed that combining anti-CD73 mAb withanti-PD-1 mAb was associated with complete responses.Consistent with previous work, the antitumor effects ofboth anti-CD73 and anti-PD-1 mAbs were dependent onIFN-g and independent of perforin (32, 43). Whether thisis an idiosyncratic feature of mouse models or whetherIFN-g is also the preferred mechanism of action in humansremains to be determined.Remarkably, the combination anti-CD73/anti-PD-1

mAbswas significantlymore effective than the combinationanti-CD73/anti-CTLA-4 mAbs, against both subcutaneous

tumors and metastatic disease. This may be due to severalnonexclusive factors. First, thismay be a consequence of thesuperior antitumor activity of anti-PD-1 versus anti-CTLA-4mAb, when administered as single agents. Second, it couldbe due to synergistic effects of anti-CD73 and anti-PD-1mAbs on Tregs. Accordingly, we have previously shown thatFoxp3þ Tregs heavily rely on CD73 to promote MC38-OVAtumor growth (28) and several studies showed that PD-1/PD-L1 interactions are important for differentiation ofinducible Foxp3þ Tregs (44, 45). Thus, the combinationanti-CD73/anti-PD-1 therapy might effectively abrogatetumor-promoting effects of Tregs. Alternatively, anti-CD73mAb might block nonenzymatic functions of CD73 (T-celladhesion for instance), which might potentiate immunecheckpoint blockade. The mechanism behind anti-CD73/anti-PD-1 mAb combination thus remains unclear andrequires further investigation.

In summary, the relatively low complete response ratescurrently observed in clinical trials with immune check-point inhibitors as single-agent therapy highlights theimportance of new combinatorial treatments able tostrike an optimal balance between tumor immunity andautoimmunity. The production of extracellular adenosineby CD73 has recently been shown to be an importantimmunosuppressive pathway contributing to tumorimmune evasion (24–26). Because extracellular adeno-sine accumulates in hypoxic tumors (46, 47), we believethat targeting this pathway will synergize with immunecheckpoint inhibitors. As was recently shown (48), com-bined blockade of nonredundant immunosuppressivepathways, i.e. indoleamine 2,3-dioxygenase (IDO) andCTLA-4, can induce potent synergistic antitumor activity.Similar to IDO, CD73-derived extracellular adenosine is apotent suppressor of effector T cells in tumors, especiallythose associated with Th1 responses. Our study suggeststhat targeted blockade of CD73 is another promisingstrategy to enhance the therapeutic activity of CTLA-4and PD-1 checkpoint inhibition. In conclusion, it is ourcontention that targeted blockade of the immunosup-pressive effects of extracellular adenosine may constitutean effective means to enhance the antitumor activity ofimmune checkpoint inhibitors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed by the authors.

Authors' ContributionsConception and design: M.J. Smyth, J. StaggDevelopment of methodology: S. Pommey, J. StaggAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): B. Allard, M.J. Smyth, J. StaggAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): B. Allard, M.J. Smyth, J. StaggWriting, review, and/or revisionof themanuscript:B.Allard,M.J. Smyth,J. StaggAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Pommey, J. StaggStudy supervision: J. Stagg

AcknowledgmentsJS was supported by a Canadian Institutes of Health Research (CIHR)

operating grant and the Famille Jean-Guy Sabourin Research Chair of






Universit�e de Montr�eal. This research was supported in part by a grant fromSusan G. Komen for the Cure. MJS was supported by a National Health andMedical Research Council (NH&MRC) Australia Fellowship and NH&MRCProgram Grant.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 26, 2013; revised August 1, 2013; accepted August 20,2013; published OnlineFirst August 27, 2013.

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2013;19:5626-5635. Published OnlineFirst August 27, 2013.Clin Cancer Res Bertrand Allard, Sandra Pommey, Mark J. Smyth, et al. Anti-CTLA-4 mAbsTargeting CD73 Enhances the Antitumor Activity of Anti-PD-1 and

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