anti-ox40 antibody directly enhances the function of tumor-reactive … · tumor-reactive cd8 þ t...

Translational Cancer Mechanisms and Therapy

Anti-OX40 Antibody Directly Enhances TheFunction of Tumor-Reactive CD8þ T Cells andSynergizes with PI3Kb Inhibition in PTEN LossMelanomaWeiyi Peng1, Leila J.Williams1, Chunyu Xu1, Brenda Melendez1, Jodi A. McKenzie1,Yuan Chen1, Heather L. Jackson2, Kui S. Voo3, Rina M. Mbofung1, Sara Elizabeth Leahey1,Jian Wang4, Gregory Lizee1, Hussein A. Tawbi1, Michael A. Davies1, Axel Hoos2,James Smothers2, Roopa Srinivasan2, Elaine M. Paul2, Niranjan Yanamandra2, andPatrick Hwu1

Abstract

Purpose: OX40 agonist–based combinations are emergingas a novel avenue to improve the effectiveness of cancerimmunotherapy. To better guide its clinical development, wecharacterized the role of the OX40 pathway in tumor-reactiveimmune cells. We also evaluated combining OX40 agonistswith targeted therapy to combat resistance to cancer immu-notherapy.

Experimental Design: We utilized patient-derived tumor-infiltrating lymphocytes (TILs) and multiple preclinical mod-els to determine the direct effect of anti-OX40 agonistic anti-bodies on tumor-reactive CD8þ T cells. We also evaluated theantitumor activity of an anti-OX40 antibody plus PI3Kbinhibition in a transgenic murine melanoma model (Brafmutant, PTEN null), which spontaneously develops immu-notherapy-resistant melanomas.

Results: We observed elevated expression of OX40 intumor-reactive CD8þ TILs upon encountering tumors;

activation of OX40 signaling enhanced their cytotoxicfunction. OX40 agonist antibody improved the antitumoractivity of CD8þ T cells and the generation of tumor-specific T-cell memory in vivo. Furthermore, combininganti-OX40 with GSK2636771, a PI3Kb-selective inhibitor,delayed tumor growth and extended the survival of micewith PTEN-null melanomas. This combination treatmentdid not increase the number of TILs, but it insteadsignificantly enhanced proliferation of CD8þ TILs andelevated the serum levels of CCL4, CXCL10, and IFNg ,which are mainly produced by memory and/or effector Tcells.

Conclusions: These results highlight a critical role ofOX40 activation in potentiating the effector function oftumor-reactive CD8þ T cells and suggest further evaluationof OX40 agonist–based combinations in patients withimmune-resistant tumors.

IntroductionSeveral immunomodulatory agents that target T-cell coinhibi-

tory receptors, such as PD-1 and CTLA-4, have been developed toboost T-cell–mediated antitumor immune responses in patientswith cancer. These immunotherapies have demonstrated durableclinical benefit in many types of cancer, and immune checkpointblockade has become a standard first-line treatment in multiplesolid cancers, including melanoma, lung cancer, bladder cancer,and kidney cancer (1, 2). This new clinical paradigm has shiftedresearch efforts in tumor immunology to prioritize the identifi-cation of additional immunoregulatory targets and rational com-binatorial treatments to further increase the rate of potent anddurable antitumor immune responses.

T-cell activation is tightly regulated by two sets of signals via T-cell receptors (TCRs) and T-cell cosignaling receptors. Positive(costimulatory) and negative (coinhibitory) signals from T-cellcosignaling receptors direct T-cell function in response to TCRstimulation. Several studies have demonstrated that activating T-cell costimulatory receptors, such as OX40 and 4-1BB, can facil-itate T-cell–mediated antitumor immunity (3, 4). Moreover,disrupting T-cell coinhibitory signaling pathways, such as PD-1and CTLA-4, has been reported to reinvigorate tumor-reactive T

1Department of Melanoma Medical Oncology, The University of Texas MDAnderson Cancer Center, Houston, Texas. 2Oncology R&D, Immuno-Oncologyand Combinations RU, GlaxoSmithKline, Collegeville, Pennsylvania. 3Depart-ment of Oncology Research for Biologics and Immunotherapy TranslationPlatform, The University of Texas MD Anderson Cancer Center, Houston, Texas.4Department of Biostatistics, The University of Texas MD Anderson CancerCenter, Houston, Texas.

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

Current address forW. Peng andC.Xu:Department ofBiologyandBiochemistry,University of Houston, Houston, Texas; current address for J.A. McKenzie, EisaiInc., Woodcliff Lake, New Jersey; and current address for R.M. Mbofung, MerckResearch Laboratories, Palo Alto, California.

Corresponding Authors: Patrick Hwu, The University of Texas MD AndersonCancer Center, 1515 Holcombe Blvd Unit 421, Houston, TX 77030. Phone: 713-792-4906; Fax: 713-745-1046; E-mail: [email protected]; NiranjanYanamandra, Immuno-Oncology and Combinations RU, GlaxoSmithKline,1250 s, Collegeville Rd, Collegeville, PA 19426. Phone: 610-917-5123; E-mail:[email protected]; and Weiyi Peng, The University of Houston,3517 Cullen Blvd, Houston, TX 77204. Phone: 713-743-6941; Fax: 713-743-3415;E-mail: [email protected]

Clin Cancer Res 2019;25:6406–16

doi: 10.1158/1078-0432.CCR-19-1259

�2019 American Association for Cancer Research.

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cells and stem tumor development in patients with a variety oftumors (5). However, a durable and effective antitumor immuneresponse only can be achieved in a small percentage of patientswith cancer treated with immune checkpoint blockade (ICB;ref. 6). Onemechanismof primary resistance to ICB is insufficienttumor-reactive T cells in patients with nonimmunogenictumors (7). Under the notion that activation of T-cell costimu-latory signaling pathways can augment the generation of effectorand memory T cells (8), more studies are focused on targetingT-cell costimulatory receptors to overcome primary resistance toICB therapy in patients with cancer. One such prominent T-cellcostimulatory molecule is OX40. Indeed, early phase clinicaltrials evaluating agonist antibodies targeting the OX40 pathwayalone or in combination with ICB in patients with cancer areongoing, such as NCT02221960 (formerly of MedImmune),NCT02528357 (GlaxoSmithKline), and NCT02554812 (Pfizer).While these trials have begun, an improved understanding of theimpact of OX40 activation on immune effector cells may help tooptimize the clinical evaluation of OX40-based immunotherapyand develop novel combinatorial approaches to treat patientswith cancer with primary resistance to ICB.

Here, by utilizing melanoma patient-derived cell lines andmultiple preclinical models, we sought to determine the role ofthe OX40 pathway in regulating the effector function of tumor-reactiveTcells andevaluate the therapeuticpotentialof combiningOX40 agonist antibody with cancer-targeted therapy. Our resultsdescribe thevalueof anOX40agonist antibody to augmentT-cell–mediated antitumor response by directly enhancing proliferationand cytotoxicity of CD8þ tumor-reactive T cells. This study alsoprovides critical rationale for the clinical evaluation of the com-bination of an OX40 agonist antibody and a selective PI3Kbinhibitor in patients with immunoresistant PTEN loss tumors.

Materials and MethodsCell lines and mice

Human Mel2399, Mel2559, and their autologous tumor-infiltrating lymphocytes (TILs) were established from patientswithmetastatic melanoma enrolled in the adoptive T-cell therapy(ACT) trial at MD Anderson Cancer Center (MDACC, Houston,TX) as described previously (9). ThemurineMC38/gp100 cell line

was generated in our previous study (10). All tumor cell linesweremaintained in RPMI1640 complete medium supplemented with10% heat-inactivated FBS (Atlanta Biologicals) and Normocin(InvivoGen). TIL cell lines were maintained in RPMI1640 with10% human type AB Serum (GEMINI), 3,000 IU/mL IL2 (Pro-metheus Laboratories), and normocin. Cells were routinelymon-itored for Mycoplasma contamination by using the MycoAlert Kit(Lonza). Short tandem repeat profiling was used to confirm theidentity of patient-derived cell lines. Themaximum length of timeof in vitro cell culture between thawing and use in the describedexperiments was 2 weeks.

C57BL/6 mice and C57BL/6 albino mice were purchasedfrom Charles River Frederick Research Model Facility. Tyr:CreER;PTENlox/lox; BRAF V600E/þ (BP) mice bred onto a C57BL/6 back-ground were kindly provided by Dr. Bosenberg (Yale UniversitySchool of Medicine, New Haven, CT). Pmel-1 TCR/Thy1.1 micewere from in-house breeding colonies. All mice were maintainedin a specific pathogen-free barrier facility at MDACC. All studieswere conducted in accordance with the MDACC and GlaxoS-mithKline (GSK) Policy on the Care, Welfare, and Treatment ofLaboratory Animals. All animal experiment protocols werereviewed by the Institutional Animal Care and Use Committeeeither at GSK or at MDACC, the institution where the work wasperformed.

Caspase-3–based T-cell killing assayPatient-derived tumor cells were labeled with DDAO Dye

(Thermo Fisher Scientific) according to the manufacturer'sinstructions. Peripheral blood mononuclear cells (PBMCs) fromhealthy donors were isolated from buffy coats (Gulf CoastRegional Blood Center) and irradiated with 5,000 rad of gammaradiation. Irradiated PBMCs were washed with PBS and thenincubated with 10 mg/mL full length or Fc-fragment deletedanti-human OX40 (GSK3174998, GSK) at 37�C for 1 hour.Antibody-pulsed PBMCs were mixed with DDAO-labeled tumorcells and autologous TILs at 37�C for an additional 3 hours. Theratio of T cells to PBMCs used in this assay was 1:1. Toevaluate the effect of the activation of the OX40 pathway inmurine CD8þ T cells, we cross-linked anti-mouse OX40 antibodyby pretreating bone marrow–derived dendritic cells (DCs)from C57BL/6 mice with 10 mg/mL anti-murine OX40 antibodyat 37�C for 1 hour. After washing with PBS, antibody-pulsed DCswere mixed with gp100-specific CD8þ Pmel-1 T cells andMC38/gp100 tumor cells for an additional 3 hours. The ratio ofDCs to T cells was 1:1. The cell mixtures were then permeabilizedwith Fix/Perm Solution (BD Biosciences) for 20 minutesat room temperature and stained with a PE-conjugated anti-cleaved caspase-3 mAb (BD Biosciences) as described previous-ly (11). Samples were analyzed using a FACSCanto II (BDBiosciences). The percentage of cleaved caspase-3þ tumor cellswas calculated and used to determine the extent of T-cell–inducedtumor apoptosis.

Retroviral transduction of pmel-1 T cellsFull-length human OX40 was amplified and cloned into a

retroviral vector, pMXs-IG, which was kindly provided by Dr.Kitamura (University of Tokyo, Japan; ref. 12). The retroviralvector expressing an enhanced firefly luciferase was generated inour previous study (13). Retroviral vectors and the packagingvectors were transiently cotransfected into the packaging cell line,Plate-E, using Lipofectamine 2000 (Invitrogen). Supernatants

Translational Relevance

Cancer immunotherapy is revolutionizing cancer treat-ment. However, most patients still fail to respond to currentlyavailable immunomodulatory agents. Thus, there remains acritical need to identify novel immunoregulatory targets andrational combinatorial strategies to induce robust and durableantitumor immune responses. Here, we used patient samplesand clinically relevant animal models to evaluate the immu-nological and antitumor effects of OX40 agonist–basedimmunotherapy. Our results add to the growing body ofevidence that OX40 agonists can boost antitumor immuneresponses by modulating T-cell effector function and tumor-specific memory. Our results also identify a novel therapeuticstrategy of combining OX40 agonist antibodies with targetedtherapy in patients with cancer, particularly those with tumorswith loss of the tumor suppressor PTEN.

OX40 Agonist–Based Cancer Immunotherapy

www.aacrjournals.org Clin Cancer Res; 25(21) November 1, 2019 6407



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containing viral particles were used to infect preactivated spleno-cytes from pmel-1 mice as described previously (10). Three daysafter transduction, transduced pmel-1 T cells were sorted using aFACSAria (BD Biosciences) based on the expression of appropri-ate reporter genes embedded in the expression vectors.

Tumor and vaccination modelsTodetermine the in vivo effect of targetingOX40on the function

of tumor-reactive CD8þ T cells, luciferase-expressing pmel-1 Tcells were transferred into C57BL/6 albino mice bearing MC38/gp100 tumor as described previously (10). One-hundred micro-grams of anti-mouse OX40 (OX-86, BioXCell) or mouse anti-human OX40 (Kindly provided K.S. Voo; ref. 14) was intraper-itoneally administered to tumor-bearing mice (two-times perweek). Tumor size was monitored every two days, and in vivobioluminescence imaging analyses were performed by using anIVIS 200 System (Xenogen) on day 6 after T-cell transfer.

To evaluate the antitumor activity of anti-OX40 alone and incombination with PI3Kb inhibition, Tyr:CreER; PTENlox/lox;BRAF V600E/þ mice (BP mice, 6–8 weeks of age) were treated with4-hydroxytamoxifen to induce tumor formation. Tumor-bearingmicewere randomized into four groups to receive anti-OX40 and/orGSK2636771.GSK2636771 (GSK)was suspended in 1%(w/v)methylcellulose andadministered tomice daily byoral gavage at adose of 30 mg/kg. Anti-OX40 (OX-86, BioXCell) was adminis-tered at a dose of 50 mg/per mouse. The relevant solvent andcontrol rat IgG antibody (Sigma)were administered to animals inthe control group.

Because our previous study showed that CD40 agonistic anti-body can promote in vivo proliferation and activation of Tcells (15), when we evaluated the in vivo effect of anti-OX40monotherapy and in combinationwith selective PI3Kb inhibitionon antigen-specific T cells, we redesigned our vaccine model toeliminate the possible confounding effects of a CD40 antibody-containing immunoadjuvant on the antitumor activity of anOX40 agonist (11, 16). Briefly, C57BL/6 mice received 1,000na€�ve pmel-1 T cells intravenously and were vaccinated with twodistinct subcutaneous injections in each flank with 100 mL ofsaline containing 100 mg of human gp10025–33. In addition,vaccinated mice received 100,000 IU rhIL-2 protein i.p. once onthe day of vaccination and twice daily on the next 2 days andweretopically treated with 50 mg of 5% imiquimod cream (Aldara,Fougera) on the vaccination site once after each vaccination.

Luminex assay and profiling of tumor-infiltrating immune cellsSerum, spleen, and tumor tissue samples were collected from

BPmice on day 6 after treatment. Twenty-fivemicroliters of serumfrom each mouse was assayed using the MILLIPLEX mousecytokine/chemokine panels I, II, and III according to the manu-facturer's protocol (EMD Millipore). The concentration of eachcytokine/chemokine present in the serum samples was measuredusing a Luminex 200 system (LuminexCorporation). Fresh tumortissues were incubated with RPMI medium containing 1 mg/mLcollagenase and 100 mg/mL hyaluronidase (Sigma-Aldrich) at37�C for 60 minutes, and manually dissociated to generatesingle-cell suspensions. Single-cell suspensions from tumor orspleen tissues were then washed twice with staining buffer andincubated with a cocktail of antibodies targeting surface markersat 4�C for 30 minutes. Cells were then fixed and permeabilizedusing the Foxp3 Fix and Permeabilization Kit according to themanufacturer's protocol (eBioscience), and then incubatedwith a

cocktail of antibodies against intracellular markers. Stained sam-ples were analyzed with a FACSCanto II or a Helios Mass Cyt-ometer (Fluidigm). Antibody details for the flow cytometry andmass cytometry staining panels used in this study are provided inSupplementary Table S1.

Statistical analysesSummary statistics (e.g., mean and SEM) of the data are

reported. Assessments of differences in continuousmeasurementsbetween two groups were made using two-sample t test posteriorto data transformation (typically logarithmic, if necessary) orWilcoxon rank-sum test. Differences in tumor size and T-cellnumbers among several treatments were evaluated using ANOVAmodels. The Kaplan–Meiermethod and log-rank test were used tocompare survival between groups. P < 0.05 was consideredstatistically significant. Graph generation and statistical analyseswere performed usingGraphPad Prism (version 7) andR softwareprogramming language (version 3.1.0).

ResultsThe OX40 pathway plays a critical role in regulating theantitumor function of tumor-infiltrating T cells in patients withmelanoma

To determine the importance of the OX40 pathway in regu-lating the effector function of TILs in patients withmelanoma, wefirst assessed the expression of OX40 on TILs before and afterrestimulation with autologous tumors. We used two tumor-reactive TIL cell lines previously generated from patients withadvanced melanoma (11, 17). Cryopreserved TILs were thawedand cultured in fresh culture medium in the presence of IL2 for3–4 days. Revived TILs were incubated with autologous tumors atthe varying ratios of T cells to tumor cells (E:T ratio), and theexpression level of OX40 on the surface of TILs over time wasdetermined by flow cytometry analysis (Fig. 1A). The majority ofresting CD4þ TILs, but less than 10% of CD8þ TILs, expressedOX40 before restimulation. The percentage of OX40þ CD4þ andCD8þ TILs peaked at 6 hours after TCR stimulation with autol-ogous tumors. Seventy-2 hours after stimulation, the percentageof OX40þ TILs returned to baseline levels. To validate that theOX40 expression on restimulated TILs is tumor dependent, weassessed the percentage of OX40þ TILs after a 12-hour coincuba-tionwith tumors at E:T ratios ranging from0.1:1 to3:1. The resultsshowed that increasing the E:T ratio can enhance the percentage ofboth OX40þ CD4þ and CD8þ TILs (Supplementary Fig. S1A).These results demonstrate that OX40 expression on patient-derived TILs is inducible and under the control of TCR signaling.Notably, OX40 was expressed on a significant portion of CD8þ

TILs (�50%) at 6 hours after encountering autologous tumors(Fig. 1A), suggesting that activation of the OX40 pathway had thepotential to directly modulate the function of CD8þ T cells attumor sites. To test this hypothesis, we used a cytotoxicityassay based on the expression of cleaved caspase-3 in tumorcells to evaluate whether activation of the OX40 signaling canalter cytotoxicity of patient-derived TILs against autologoustumors. Given that immobilization of anti-OX40 antibody isrequired to activate the OX40 pathway in T cells (14), gamma-irradiated PBMCs were pulsed with anti-human OX40 (hOX40)antibody (GSK3174998) for 1 hour and used to stimulateTILs in the presence of autologous tumors. When comparedwith Fc-fragment–deleted anti-hOX40 antibody, full-length

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Clin Cancer Res; 25(21) November 1, 2019 Clinical Cancer Research6408




anti-hOX40–pulsed PBMCs significantly increased TIL-inducedapoptosis of tumors (Fig. 1B). In contrast, treatment with anti-hOX40–pulsed PBMCs alone had no impact on tumor apoptosis.In addition, anti-mouse OX40-pulsed DCs enhanced murinetumor apoptosis induced by tumor-reactive CD8þ T cells (Supple-mentary Fig. S1B). Although OX40 was not highly expressed onresting cytotoxicCD8þTILs,our results suggest that tumorexposureinduced the expression of OX40 on CD8þ TILs and that activationof OX40 signaling enhances the cytotoxic function of CD8þ TILs.

Activation of the OX40 pathway improves antitumor activity ofCD8þ T cells and facilitates the generation of tumor-specific T-cell memory

To characterize the in vivo effects of OX40 agonist antibody onantitumor activity of CD8þ T cells, we adoptively transferredluciferase-expressing tumor-reactive CD8þ T cells (pmel-1) intotumor-bearing mice 1 day after sublethal irradiation, which isrequired for expansion of transferred T cells. Thesemice were thentreated with either a control antibody or an anti-mouse OX40(mOX40) antibody as shown in Fig. 2A. The gp100-expressingMC38 tumors in mice treated with pmel-1 T cells grew signifi-cantly slower than those not treated with T cells (P <0.0001; Fig. 2B). Importantly, anti-mOX40 treatment significant-ly delayed tumor growth in all T-cell–treated mice (P <0.001; Fig. 2B). We also used bioluminescence imaging analysisto determine the change in tumor trafficking of transferred tumor-reactive T cells in response to anti-mOX40 treatment. Althoughthe average bioluminescence intensity at the tumor site in micetreated with anti-mOX40 was higher than the control group on

day 6 after T-cell transfer, the difference between these two groupwas not statistically significant (P ¼ 0.053; Fig. 2C).

Given that an OX40 agonist antibody has been reported toenhance the proliferation of CD4þ Th cells and suppress thefunction of T regulatory (Treg) cells (14), the OX40 agonist–enhanced antitumor activity of transferred CD8þ T cells observedin this model may have been achieved by indirect regulation viaCD4þ T cells (18). To test whether anti-OX40 treatment candirectly promote CD8þ T-cell function, we modified our murineACTmodel to ensure that theOX40 agonist antibodyonly targetedOX40-expressing transferredCD8þ T cells.Wefirst transduced full-length human OX40 (hOX40) cDNA into murine pmel-1 T cells,and adoptively transferred hOX40-expressingCD8þ pmel-1 T cellsinto tumor-bearing mice. Instead of using the mOX40 agonist, wetreated all experimentalmicewith either isotype control antibodyoranti-human OX40 antibody. Reduced tumor size was observed inmice from the anti-hOX40group as early as 2days after thefirst doseof antibody treatment (Fig. 2D). Bioluminescence imaging analysisof mice on day 6 after T-cell transfer revealed that the number oftransferred CD8þ T cells in the tumors was comparable between thecontrol and anti-hOX40 groups (Fig. 2E). Although OX40 agonistantibody treatment had limited impact on directly regulating tumortrafficking of CD8þ T cells, data from both models supports thatactivation of OX40 signaling promotes the effector function oftumor-reactive CD8þ T cells.

We then examined the impact of anti-OX40 on the generationof antigen-specific CD8þ memory T cells using a murine vaccinemodel (Fig. 3A; refs. 11, 16). The composition of adjuvants in ourpreviously describedmurine vaccinationmodel was simplified to

Figure 1.

Kinetics of OX40 expression and in vitro effect of OX40 agonist antibody on tumor-infiltrating T cells from patients with melanoma. A, Increased OX40expression on both CD4þ and CD8þ TILs upon restimulation by autologous tumor cells. Two patient-derived TIL lines, TIL2399 and TIL2559, were cultured inT-cell growth medium in the presence of IL2 for at least 3 days. Revived TILs were then cocultured with autologous tumor cells at different ratios of E:T. Thepercentage of TILs expressing OX40was determined by flow cytometry analysis at indicated the timepoints. B, Cross-linked anti-OX40 antibody enhanced thecytotoxicity of TILs against autologous tumors. Irradiated PBMCs from healthy donors were pulsed with 10 mg/mL of full-length or Fc-fragment–deleted anti-human OX40 (GSK3174998) at 37�C for 1 hour. After washing with PBS, antibody-pulsed PBMCs were mixed with DDAO-labeled tumor cells (Mel2559) andautologous TILs (TIL2559) at 37�C for an additional 3 hours. The cell mixtures were stained intracellularly with an anti-cleaved caspase-3 antibody. Thecytotoxicity of TILs against tumors was evaluated by flow cytometry analysis based on the percentage of cleaved caspase-3þ DDAO-labeled tumor cells.One-way ANOVA demonstrated statistical significance (� , P < 0.05). Representative data from three independent experiments are shown.






avoid confounding effects of the anti-CD40 antibody on anti-OX40 antibody activity. Briefly, fresh splenocytes from pmel-1mice were adoptively transferred into C57BL/6 mice. Experimen-talmicewere then vaccinatedwith the gp100peptide onday0 andday 28, and treated with anti-mOX40 on days 0, 4, 7, 12, 28, 31,35, and 40. One week after the last anti-OX40 treatment, exper-imental mice were challenged with gp100-expressing MC38tumor cells. By monitoring the percentage of transferred pmel-1 T cells in PBMCs, we found that anti-OX40 enhanced theproliferation of pmel-1 T cells after initial antigen stimulation(on day 5), and this positive effect of anti-OX40 was dosedependent (Fig. 3B). Five days after the booster vaccine (on day33), the percentage of antigen-specific T cells in peripheral bloodCD8þ T cells in mice was also significantly higher than the rest ofvaccinated mice (Fig. 3B). Moreover, administration of 200 mg ofanti-OX40 to gp100-vaccinated mice before tumor inoculation

successfully suppressed the development of gp100-expressingtumors, indicating that OX40 agonists can induce the generationof tumor-reactive memory T cells (Fig. 3C).

OX40 agonist antibody synergizes with GSK2636771 incontrolling the development of PTEN-null melanoma

Previously, we demonstrated that oncogenic activation of thePI3K pathway by PTEN loss promotes tumor-associated immu-nosuppressive mechanisms and is associated with poor clinicaloutcomes in patients with melanoma treated with anti–PD-1(11). We also found that Braf-mutant, PTEN-null melanomasdeveloped in Tyr:CreER; BRAFV600E/þ; PTENlox/lox mice (BP mice)are resistant to immune checkpoint inhibitory antibodies. How-ever, improved tumor growth inhibition was observed with thecombination of anti–PD-1 antibodies with GSK2636771, aPI3Kb-selective inhibitor, which was selected on the basis of data

Figure 2.

In vivo effect of OX40 activation on antitumor activity and memory generation of tumor-reactive T cells. A, Experimental setup of a murine ACT protocol toevaluate in vivo effect of the activation of OX40 signaling on tumor-reactive T cells. B, Increased antitumor activity of tumor-reactive T cells in the presence ofanti-mouse OX40 antibody. Pmel-1 T cells that express a TCR specifically recognizing a melanoma tumor antigen (gp100) were modified to express luciferase forin vivomonitoring of tumor trafficking. Luciferase-expressing pmel-1 T cells were transferred into mice bearing gp100-expressing MC38 tumors (N¼ 4 pergroup). All experimental mice were then treated with DC vaccine and IL2 as described previously (41). One-hundred micrograms per dose of anti-OX40 orcontrol antibody was used to treat mice twice weekly for 2 weeks. Tumor size was monitored every 2 days. C, Luciferase signaling intensity at tumor sites in micewith ACT. Tumor tracking of transferred pmel-1 T cells was evaluated on day 6 after T-cell transfer. Quantitative imaging analysis revealed that anti-mOX40 didnot significantly alter tumor tracking of transferred T cells. D, Anti-human OX40 antibody facilitated human OX40-expressing pmel-1 T cells control of the growthof MC38/gp100 tumors (N¼ 5 per group). E, In vivo tumor tracking of human OX40-expressing T cells in response to anti-human OX40 antibody treatment.Quantitative imaging analysis revealed that anti-hOX40 did not significantly alter tumor trafficking of transferred T cells. The pairwise multiple comparisons aftertwo-way ANOVA test and the t test were used to evaluate the statistical significance of the difference in tumor growth and tumor trafficking, respectively.�� , P < 0.001 and �� , P < 0.0001. Representative data from two independent experiments are shown.

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supporting a role for this PI3K isoform–selective inhibition incells with loss of PTEN (11, 19, 20). The combination of pem-brolizumab and GSK2636771 is now being evaluated in a phaseI/II clinical trial (NCT03131908).

To evaluate the effectiveness of combining T-cell costimulatoryreceptor-based immunotherapy and targeted therapy, we com-

bined anti-OX40withGSK2636771 in the BPmodel (Fig. 4A). BPmice bearing measurable melanoma lesions were randomizedand treated with isotype control antibody, GSK2636771, anti-OX40 antibody, or a combination of the two agents. Single-agentanti-OX40 and single-agent GSK2636771 both failed to signifi-cantly inhibit tumor growth, but the combination was highly

Figure 3.

In vivoOX40 agonist antibody treatment enhanced the proliferation of tumor-reactive T cells upon TCR stimulation and induced the generation of tumor-specificT-cell memory. A, Schematic representation of a murine vaccine model used to evaluate the in vivo effect of the activation of OX40 signaling on tumor-reactiveT cells. C57BL/6 mice were transferred with the splenocytes from pmel-1 mice and vaccinated with gp100 peptide. Vaccinated mice received either controlantibody or anti-mouse OX40 antibody. After 4 weeks, mice received a second gp100 peptide vaccine (booster). Gp100-expressing MC38 tumors weresubcutaneously injected into vaccinated mice on day 47. B, The percentage of gp100-specific T cells in CD8þ T cells in the peripheral blood of mice treated withOX40 agonist antibody. Thy1.1, a congenic marker for transferred pmel-1 T cells, was used to determine the number of gp100-specific T cells in peripheral bloodafter antigen stimulation. The pairwise multiple comparisons after two-way ANOVA test demonstrated statistical significance (� , P < 0.05): control/a-OX4050 mg versus a-OX40 100 mg/a-OX40 200 mg on day 5; and control/a-OX40 50 mg versus a-OX40 200 mg on day 33. C, The growth curves of MC38/gp100tumors in vaccinated mice treated with anti-OX40 (N¼ 8 per group). Representative data from two independent experiments are shown.

Figure 4.

OX40 agonist antibody synergized with PI3Kb-selective inhibition to control the growth of PTEN-loss tumors. A, The treatment schedule of antibody and thePI3Kb inhibitor (GSK2636771) is shown. Melanomawas induced in a group of Tyr:CreER; PTENlox/lox; BRAFV600E/þmice. Mice with measureable tumors wererandomized and treated with control, GSK2636771 (30 mg/kg/day), anti-mouse OX40 (50 mg/dose), and the combination of both reagents. B, Tumor size wasmonitored in each of the treatment groups every 2 days. The pairwise multiple comparisons after two-way ANOVA test were used to determine statisticalsignificance. � , P < 0.05. C, Kaplan–Meier survival curves of mice treated with GSK2636771 and/or anti-mouse OX40. Log-rank test demonstrated statisticalsignificance (P < 0.05): GSK2636771þanti-OX40 versus control, GSK2636771, and anti-OX40 (N¼ 4–7). Data presented are a summary of two independentexperiments.






effective andmarkedly extended the survival of BP tumor-bearingmice (the median survival times of control, GSK2636771, anti-OX40, and combination groups are 14.5, 18, 14, and 30 days,respectively; P ¼ 0.0021; Fig. 4B and C). A linear mixed mod-el (21) determined that the antitumor effect of GSK2636771withanti-OX40 was synergistic (P ¼ 0.0004; Supplementary Fig. S2).

Importantly, no overt adverse effects or toxicities were observedwith the combination treatment. We further tested whetherGSK2636771, anti-OX40, or the combination affected the pro-liferation of antigen-specific T cells upon in vivo antigen stimu-lation. These experiments showed that the combination treat-ment did not significantly reduce whole-blood cell counts orinhibit the proliferation of gp100-specific T cells upon gp100peptide immunization (Supplementary Fig. S3). Taken together,these data suggest that combining anti-OX40 with GSK2636771is another potentially effective strategy to overcome immuneresistance in melanomas with PTEN loss.

Combining anti-OX40 with a PI3Kb inhibitor enhances T-cell–mediated antitumor immune activity

We then explored the underlying mechanisms by which PI3Kbinhibition synergizes with anti-OX40 to control Braf-mutant,PTEN-null melanomas. Additional tumor-bearing BP mice weretreated with anti-OX40 and/or GSK2636771 as described above.On day 6, serum, spleen, and tumor tissue samples were collectedfor immuneprofiling.Wemeasured the serumconcentrations of abroad set of chemokines/cytokines in each experimental mouseusing Luminex assays. Among 43 tested chemokines/cytokines,the combination treatment significantly increased the serumconcentrations of CCL2, CCL4, CCL15, CXCL10, and G-CSF incomparisonwith themonotherapy or control treatments (Fig. 5A;Supplementary Fig. S4). Two of these factors, CCL4 and CXCL10,are mainly produced by memory and/or effector T cells. Inaddition, the serum levels of IFNg , another important antitumorcytokine produced by T cells, were significantly higher in mice

Figure 5.

OX40 agonist antibody in combination with PI3Kb-selective inhibition altered the immune cell profile and the serum concentration of cytokine/chemokinesproduced by T cells. Tyr:CreER; PTENlox/lox; BRAFV600E/þmice with measureable tumors were treated with control, GSK2636771, anti-mouse OX40, and thecombination of both reagents. Mice were euthanized on day 6 after treatment and used to characterize the changes in the immune profile of mice in the differenttreatment groups. A, The serum levels of T-cell–associated cytokines/chemokines. Serum from each experimental mouse was collected and used to measure theconcentration of 43 cytokines/chemokines using the MILLIPLEX MAPmouse cytokine/chemokine panels. The average of the serum cytokine/chemokineconcentration in each group is shown. � , P < 0.05; �� , P < 0.01; �� , P < 0.0001 (N¼ 3). B, The results of CyTOF analysis revealed the systemic effects of anti-OX40 alone or in combination with PI3Kbi. Spleens were collected frommice in the different treatment groups and processed into single-cell suspensions at theconcentration of 20 million cells/mL. Equal amounts of single-cell suspensions from experimental mice in each group were pooled (N¼ 3). Pooled samples ofcontrol, anti-OX40, and the combination groups were analyzed by CyTOF to determine the percentages of different immune cell subsets and their proliferation(measured by Ki-67 expression). High-dimensional visualization of changes in Ki-67 expression in response to treatment was generated using SPADE. The ratioof Ki-67 (OX40 alone or combination group): Ki-67 (control group) is represented by the color scale, with blue indicating a reduced level of Ki-67 after treatment.The number and size of nodules in each immune cell subset represents the percentage of the indicated immune cell subset in spleens. Data presented are asummary of two independent experiments.

Peng et al.





who received the combination treatment than in mice treatedwith the control antibody or PI3Kb inhibitor alone (Fig. 5A).Next, we characterized the function and phenotype of immunecells in spleens frommice in the different treatment groups using a24-channel mass cytometry (CyTOF) panel (SupplementaryTable S1). High-dimensional analysis using SPADE was per-formed to examine the changes in immune cells in the treatmentgroups. Anti-OX40 monotherapy reduced the percentage of M2macrophages, which are immunosuppressive immune cellsfound in peripheral lymphoid organs. In comparison withanti-OX40monotherapy, combination treatment further reducedthe percentage ofM2macrophages and significantly increased theexpression of Ki-67 in T cells, DCs, andM1macrophages, suggest-ing enhanced proliferation of antitumor immune cells (Fig. 5B).Because of poor tumor infiltration of immune cells in this tumormodel, the number of immune cells at the tumor site wasinsufficient to perform CyTOF analysis. Therefore, we evaluatedthe changes of T-cell compartments at the tumor sites by a five-channel flow cytometry panel (Supplementary Table S1).Although GSK2636771 significantly increased the number ofCD8þ T cells within tumors, there was no significant differencein the number of tumor-infiltrating CD8þ T cells in theGSK2636771 monotherapy group versus the combination treat-ment group (Fig. 6A). In addition, neither monotherapy treat-ment nor the combination significantly altered the total numberof tumor-infiltrating CD4þ T cells (Fig. 6A). In addition, thenumber of Tregs in the tumors from the combination treatmentgroup was comparable with those of the monotherapy-treatedtumors (Fig. 6B). By using the expression of Ki-67 to determineT-cell function at tumor sites, we observed that a significantincrease in the percentage of Ki-67þ CD8þ T cells, but not in thepercentage of Ki-67þ CD4þ T cells, was detected with the com-bination versus each of the other treatment groups (Fig. 6C).These results suggest that combining GSK2636771 with anti-OX40 promotes T-cell–mediated antitumor immune responsesby inducing robust proliferation of CD8þ tumor-infiltratingT cells.

DiscussionIn this article, we examined the expression of OX40 on TILs

derived from patients with melanoma and tested whether stim-ulating OX40 signaling can promote cytotoxicity of TILs againstautologous tumor cells. Data from preclinical tumor modelsconfirmed that OX40 agonist antibody can improve T-cell–mediated antitumor immune responses by OX40 receptorengagement on CD8þ T cells and inducing a protective tumor-specific T-cell memory. To develop effective therapeuticapproaches in patients with cancer who fail to respond to ICB,we used a transgenic Braf-mutant and PTEN-loss murine model,which can spontaneously develop immune-resistant melanomas,to evaluate the efficacy of combiningOX40 agonist antibodywithtargeted therapy. The combination of an OX40 agonist antibodyand a selective PI3Kb inhibitor successfully potentiated the pro-liferation of antitumor immune cells and suppressed tumordevelopment in mice bearing Braf-mutant and PTEN-lossmelanoma.

OX40, also known as TNFRSF4 or CD134, belongs to the TNFreceptor superfamily (22). The engagement of three molecules ofOX40 and trimeric OX40 ligand (OX40L) initiates the signalingcascade through TNF receptor–associated factors and eventuallydrives NF-kB activation (8). Although OX40 expression can beinduced by TCR activation in both CD4þ and CD8þ T cells, theexpression of OX40 in CD4þ TILs is significantly higher than inCD8þ TILs (23, 24). In addition, in vitro and in vivo studies usingviral infection and autoimmune disease models have demon-strated that the effect ofOX40 activation onCD8þ T cells is largelyindirect and is mediated by OX40 regulation of CD4þ Th cellfunction (25–27). The activation of the OX40 pathway in regu-latory CD4þ T (Treg) cells has also been reported to blunt theimmunosuppressive function of Treg cells (14). Therefore, thecurrent working model of OX40 agonist antibody function intumorsmainly focuses on its effect onCD4þT cells. In our studies,we evaluated the OX40 expression levels in established TIL linesfrom patients with advanced melanoma under different culture

Figure 6.

OX40 agonist antibody in combination with PI3Kb-selective inhibition promoted the proliferation of tumor-infiltrating CD8þ T cells in mice with PTEN-losstumor. Tyr:CreER; PTENlox/lox; BRAFV600E/þmice with measureable tumors were treated with control, GSK2636771, anti-mouse OX40, and the combination ofboth reagents (N¼ 3). On day 6 after treatment, tumor tissues were harvested, weighted, and stained with antibodies for flow cytometry analysis.A, The totalnumber of CD4þ and CD8þ T cells in tumors frommice treated with anti-OX40 and/or GSK2636771. B, The percentage of Treg cells (CD25þFOXp3þ) in CD4þ Tcells in tumors frommice treated with anti-OX40 and/or GSK2636771. C, The percentage of Ki-67þ CD4þ and CD8þ T cells in tumors frommice treated with anti-OX40 and/or GSK2636771. One-Way ANOVA test demonstrated statistical significance (P < 0.05): � , P < 0.05; and �� , P < 0.01. Data presented are a summary oftwo independent experiments.






conditions. Consistent with the results fromother types of cancer,OX40 is predominantly expressed on resting CD4þ TILs. How-ever, upon encountering autologous tumor cells, the OX40expression on CD8þ TILs is significantly upregulated and isrestored to the baseline 72 hours after TCR stimulation.These results prompted us to test whether OX40 agonist antibodycan directly potentiate the function of tumor-reactive CD8þ TILs.

This hypothesis was first supported by data demonstrating thatin vitro, anti-OX40 antibody can promote the proliferation ofna€�ve CD8þ T cells after anti-CD3 stimulation (14). In our study,by using patient-derived TILs and paired autologous tumor cells,we found that cross-linked OX40 agonist antibody facilitatedtumor apoptosis induced by autologous TILs in vitro. Given thatthe tumor-specific cytotoxicity of TILs used in this study has beenpreviously shown to be largely dependent on the expression ofMHC class I molecules (11), our results suggest that OX40signaling can directly regulate cytotoxicity of tumor-reactiveCD8þ TILs. In addition, we examined the changes in CD8þ

T cells in response to anti-OX40 treatment usingmultiple murinetumor models. Our in vivo data further demonstrated that anti-OX40 treatment enhances antitumor activity of tumor-reactiveCD8þ T cells. Furthermore, we used a vaccination model estab-lished in our previous studies to evaluate T-cell memory forma-tion (16) and found that anti-OX40 treatment promoted CD8þ

T-cell–mediated antitumor memory induced by antigen vaccina-tion. In particular, when treating tumor-bearing mice with anti-human OX40 antibody without cross-reactivity to mouse OX40,we consistently observed improved tumor suppression by humanOX40–expressing CD8þ T cells. These results imply that the directrole of OX40 signaling in tumor-reactive CD8þ T cells should notbe overlooked.

In clinic, the potential of OX40 as a target for cancer immu-notherapy was initially tested by using a murine anti-humanOX40 IgG1 mAb, 9B12 (28). Although no patients achieved aclinical response based on the RECIST, 12 of 30 treated patientshad at least one regressed metastatic nodule. Multiple fullyhuman or humanized OX40 agonist antibodies have been gen-erated in the last 2 decades. At least five different antibodies haveentered clinical development, including GSK3174998 (GSK),INCAGN01949 (Agenus), MEDI0562 (formerly of MedIm-mune), MOXR0916 (Genentech), and PF-04518600 (Pfizer;refs. 29–33). Similar to the preclinical results frommurine tumormodels (34), the early data from two phase I clinical trials showedanti-OX40 monotherapy was well tolerated in patients withcancer, with only one serious treatment-related grade 3 adverseevent (pneumonitis responsive to corticosteroids) of 71 patientsreported (29, 30). These results suggest that OX40 agonist anti-body treatment in patients with cancer is generally well-tolerated.In addition, up to 200 mg of anti-OX40 per dose for 2 weeks hadno adverse effect on the health and well-being of experimentalmice in this study.

The antitumor effect of OX40 agonist antibody in patients withcancer has not been fully elucidated. However, primary andacquired resistance to anti-OX40 monotherapy are expected inpatients with cancer due to a wide variety of tumor-associatedimmunosuppressive factors. Therefore, combining anti-OX40therapy with other treatments targeting these tumor-associatedimmunosuppressive factors may result in better response ratesand improved overall survival in patientswith cancer. In addition,eradication of well-established tumors has been reported in micetreated with anti-OX40 in combination with other immune

reagents, such as TLR9 agonist in a spontaneous breast cancermurine model (35). However, when evaluating the combinationof anti-OX40 and anti–PD-1, two research groups independentlydemonstrated that concurrent treatment using these two agentsinduced T-cell apoptosis and produced antagonistic antitumorresponses. Enhanced antitumor effect was only observed in micesequentially treated with anti-OX40 and anti–PD-1 (36, 37). Asimilar potentially antagonistic antitumor effect has beenobserved with the combination of immunotherapy and targetedtherapy. In that, although both CpG-based tumor vaccine andBRAF inhibitor have therapeutic benefit as monotherapies inpatientswith cancer, combiningCpGwith BRAF inhibitor negatesthe antitumor effect of BRAF inhibitor in Braf-mutant tumors in aB-cell–dependent manner (38). Therefore, to develop potenttherapeutic strategies for OX40-based cancer treatment, we needto not only rationally choose combination partners with com-plementing effects, but also optimize treatment schedules tomaximize the antitumor effect of anti-OX40.

Melanomas that spontaneously develop in transgenic micebearing the Braf V600E mutation and PTEN loss in melanocytes,display primary resistance to cancer immunotherapy, due to lackof tumor-associated antigens and upregulated immunosuppres-sive factors induced by oncogenic activation of the PI3K path-way (11, 39, 40). In this study, we used this immune-resistanttumor model to evaluate the therapeutic efficacy of anti-OX40 incombination with PI3K inhibition. Although anti-OX40 mono-therapy did not effectively control tumor development, concur-rent treatment of anti-OX40 and PI3Kb-selective inhibition sig-nificantly delayed tumor growth and extended the survival ofmice bearing Braf-mutant and PTEN-loss melanomas. Unlike thecombination of PI3Kb inhibition and ICB, this combinatorialapproach did not significantly increase the number of CD4þ andCD8þ T cells at the tumor sites but promoted the proliferation ofCD8þ T cells at the tumor sites. Anti-OX40 plus PI3Kb inhibitortreatment also systemically enhanced the proliferation of antitu-mor immune cells but reduced the number of immunosuppres-sive M2 macrophages. We also found elevated serum levels ofcytokine/chemokines, which were predominantly produced byeffector T cells in mice treated with the combination therapy. Inaddition, our studies showed that this combination did notincrease the susceptibility of T cells to activation-induced apo-ptosis. Overall, our results offer the first preclinical evidencedemonstrating that combining anti-OX40 with PI3Kb inhibitorcould be an effective treatment for patients with PTEN-losstumors. These results also provide the rationale to clinically testthis combination in patients with immunoresistant PTEN-losstumors.

Taken together, our studies suggest that OX40 agonist–basedcombination treatment can induce a robust and durable antitu-mor immune response by promoting effector T-cell function andthe generation of memory T cells.

Disclosure of Potential Conflicts of InterestW. Peng reports receiving speakers bureau honoraria from Bristol-Myers

Squibb and reports receiving commercial research grants fromGlaxoSmithKline. H. Jackson is an employee of and has ownership interests(including patents) at GlaxoSmithKline. K.S. Voo is listed as a co-inventoron a patent on Anti-OX40 antibody for the treatment of cancer which isowned by MD Anderson Cancer Center and licensed to GlaxoSmithKline.H.A. Tawbi is a consultant/advisory board member for Bristol-MyersSquibb, Novartis, Merck, Genentech and Array, and reports receivingcommercial research grants from Bristol-Myers Squibb, Merck, Genentech,

Peng et al.





GlaxoSmithKline and Celgene. M.A. Davies is a consultant/advisory boardmember for Novartis, GlaxoSmithKline, Roche Genentech, Bristol-MyersSquibb, Sanofi Aventis, NanoString, and reports receiving commercialresearch grants from GlaxoSmithKline, Bristol-Myers Squibb, Roche/Genentech, Sanofi Aventis, AstraZeneca and Oncothyreon. A. Hoos is anemployee of and has ownership interests (including patents) atGlaxoSmithKline and Imugene, and is a consultant/advisory board memberfor CRI. R. Srinivasan has ownership interests (including patents) atGlaxoSmithKline. E.M. Paul is an employee of and has ownership interests(including patents) at GlaxoSmithKline. N. Yanamandra is an employeeof and has ownership interests (including patents) at GlaxoSmithKline.P. Hwu has ownership interests (including patents) at Dragonfly andImmatics, is a consultant/advisory board member for Dragonfly, Immatics,GlaxoSmithKline and Sanofi. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: W. Peng, K.S. Voo, J. Smothers, E.M. Paul,N. Yanamandra, P. HwuDevelopment of methodology: W. Peng, K.S. Voo, P. HwuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. Peng, L.J. Williams, C. Xu, B. Melendez,J.A. McKenzie, Y. Chen, K.S. Voo, R.M. Mbofung, S.E. LeaheyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. Peng, C. Xu, B. Melendez, H. Jackson, K.S. Voo,J. Wang, H.A. Tawbi, M.A. Davies, A. Hoos, N. Yanamandra, P. HwuWriting, review, and/or revision of the manuscript: W. Peng, J.A. McKenzie,K.S. Voo, J.Wang, G. Lizee, H.A. Tawbi,M.A. Davies, A. Hoos, E.M. Paul, P. Hwu

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):W. Peng, L.J. Williams, N. Yanamandra, P. HwuStudy supervision: W. Peng, G. Lizee, J. Smothers, R. Srinivasan

AcknowledgmentsThis work was supported in part by the following NCI grants: R01CA187076

(to P. Hwu and M.A. Davies), P50CA093459 (to UT M.D. Anderson CancerCenter SPORE in Melanoma), T32CA009666-21(to M.A. Davies), andP30CA016672 (to UT MDACC CCSG for the Flow Cytometry & CellularImaging facility), by philanthropic contributions to MDACCMelanoma MoonShots Program; Melanoma Research Alliance Young Investigator Award (toW. Peng, 558998); Dr. Miriam and Sheldon G. Adelson Medical ResearchFoundation; Aim at Melanoma Foundation, Miriam and Jim Mulva ResearchFund; and by Cancer Prevention and Research Institute of Texas (to P. HwuRP170401 and to J.A. McKenzie RP140106 and RP170067).

The authors would like to thank the past and present TIL lab members atMDACC:Orenthial J. Fulbright, ArelyWahl, Esteban Flores, Shawne T. Thorsen,Ren�e J. Tavera, Renjith Ramachandran, Audrey M. Gonzalez, ChristopherToth, SethWardell, and RahmatuMansaray as well as Drs. Chantale Bernatchez,Cara Haymaker, Marie-Andr�ee Forget, and Shruti Malu for generation ofresearch TIL and tumor lines.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 21, 2019; revised June 10, 2019; accepted July 26, 2019;published first August 1, 2019.

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2019;25:6406-6416. Published OnlineFirst August 1, 2019.Clin Cancer Res Weiyi Peng, Leila J. Williams, Chunyu Xu, et al. in PTEN Loss Melanoma

Inhibitionβ T Cells and Synergizes with PI3K+Tumor-Reactive CD8Anti-OX40 Antibody Directly Enhances The Function of

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