Anti-OX40 Antibody Directly Enhances the Function of Tumor-Reactive Cd8þ T Cells and Synergizes with Pi3kb Inhibition in PTEN Loss Melanoma Weiyi Peng1, Leila J

Published OnlineFirst August 1, 2019; DOI: 10.1158/1078-0432.CCR-19-1259

Translational Cancer Mechanisms and Therapy Clinical Cancer Research Anti-OX40 Antibody Directly Enhances The Function of Tumor-Reactive CD8þ T Cells and Synergizes with PI3Kb Inhibition in PTEN Loss Melanoma Weiyi 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, and Patrick Hwu1

Abstract

Purpose: OX40 agonist–based combinations are emerging activation of OX40 signaling enhanced their cytotoxic as a novel avenue to improve the effectiveness of cancer function. OX40 agonist antibody improved the antitumor þ immunotherapy. To better guide its clinical development, we activity of CD8 T cells and the generation of tumor- characterized the role of the OX40 pathway in tumor-reactive specific T-cell memory in vivo.Furthermore,combining immune cells. We also evaluated combining OX40 agonists anti-OX40 with GSK2636771, a PI3Kb-selective inhibitor, with targeted therapy to combat resistance to cancer immu- delayed tumor growth and extended the survival of mice notherapy. with PTEN-null melanomas. This combination treatment Experimental Design: We utilized patient-derived tumor- did not increase the number of TILs, but it instead þ infiltrating lymphocytes (TILs) and multiple preclinical mod- significantly enhanced proliferation of CD8 TILs and els to determine the direct effect of anti-OX40 agonistic anti- elevated the serum levels of CCL4, CXCL10, and IFNg, þ bodies on tumor-reactive CD8 T cells. We also evaluated the which are mainly produced by memory and/or effector T antitumor activity of an anti-OX40 antibody plus PI3Kb cells. inhibition in a transgenic murine melanoma model (Braf Conclusions: These results highlight a critical role of mutant, PTEN null), which spontaneously develops immu- OX40 activation in potentiating the effector function of þ notherapy-resistant melanomas. tumor-reactive CD8 T cells and suggest further evaluation Results: We observed elevated expression of OX40 in of OX40 agonist–based combinations in patients with þ tumor-reactive CD8 TILs upon encountering tumors; immune-resistant tumors.

1Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 2Oncology R&D, Immuno-Oncology Introduction 3 and Combinations RU, GlaxoSmithKline, Collegeville, Pennsylvania. Depart- Several immunomodulatory agents that target T-cell coinhibi- ment of Oncology Research for Biologics and Immunotherapy Translation Platform, The University of Texas MD Anderson Cancer Center, Houston, Texas. tory receptors, such as PD-1 and CTLA-4, have been developed to – 4Department of Biostatistics, The University of Texas MD Anderson Cancer boost T-cell mediated antitumor immune responses in patients Center, Houston, Texas. with cancer. These immunotherapies have demonstrated durable fi Note: Supplementary data for this article are available at Clinical Cancer clinical bene t in many types of cancer, and immune checkpoint Research Online (http://clincancerres.aacrjournals.org/). blockade has become a standard first-line treatment in multiple solid cancers, including melanoma, lung cancer, bladder cancer, Current address for W. Peng and C. Xu: Department of Biology and Biochemistry, University of Houston, Houston, Texas; current address for J.A. McKenzie, Eisai and kidney cancer (1, 2). This new clinical paradigm has shifted Inc., Woodcliff Lake, New Jersey; and current address for R.M. Mbofung, Merck research efforts in tumor immunology to prioritize the identifi- Research Laboratories, Palo Alto, California. cation of additional immunoregulatory targets and rational com- Corresponding Authors: Patrick Hwu, The University of Texas MD Anderson binatorial treatments to further increase the rate of potent and Cancer Center, 1515 Holcombe Blvd Unit 421, Houston, TX 77030. Phone: 713- durable antitumor immune responses. 792-4906; Fax: 713-745-1046; E-mail: [email protected]; Niranjan T-cell activation is tightly regulated by two sets of signals via T- Yanamandra, Immuno-Oncology and Combinations RU, GlaxoSmithKline, cell receptors (TCRs) and T-cell cosignaling receptors. Positive 1250 s, Collegeville Rd, Collegeville, PA 19426. Phone: 610-917-5123; E-mail: (costimulatory) and negative (coinhibitory) signals from T-cell [email protected]; and Weiyi Peng, The University of Houston, cosignaling receptors direct T-cell function in response to TCR 3517 Cullen Blvd, Houston, TX 77204. Phone: 713-743-6941; Fax: 713-743-3415; E-mail: [email protected] stimulation. Several studies have demonstrated that activating T- cell costimulatory receptors, such as OX40 and 4-1BB, can facil- Clin Cancer Res 2019;XX:XX–XX itate T-cell–mediated antitumor immunity (3, 4). Moreover, doi: 10.1158/1078-0432.CCR-19-1259 disrupting T-cell coinhibitory signaling pathways, such as PD-1 2019 American Association for Cancer Research. and CTLA-4, has been reported to reinvigorate tumor-reactive T

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was generated in our previous study (10). All tumor cell lines were Translational Relevance maintained in RPMI1640 complete medium supplemented with Cancer immunotherapy is revolutionizing cancer treat- 10% heat-inactivated FBS (Atlanta Biologicals) and Normocin ment. However, most patients still fail to respond to currently (InvivoGen). TIL cell lines were maintained in RPMI1640 with available immunomodulatory agents. Thus, there remains a 10% human type AB Serum (GEMINI), 3,000 IU/mL IL2 (Pro- critical need to identify novel immunoregulatory targets and metheus Laboratories), and normocin. Cells were routinely mon- rational combinatorial strategies to induce robust and durable itored for Mycoplasma contamination by using the MycoAlert Kit antitumor immune responses. Here, we used patient samples (Lonza). Short tandem repeat profiling was used to confirm the and clinically relevant animal models to evaluate the immu- identity of patient-derived cell lines. The maximum length of time nological and antitumor effects of OX40 agonist–based of in vitro cell culture between thawing and use in the described immunotherapy. Our results add to the growing body of experiments was 2 weeks. evidence that OX40 agonists can boost antitumor immune C57BL/6 mice and C57BL/6 albino mice were purchased responses by modulating T-cell effector function and tumor- from Charles River Frederick Research Model Facility. Tyr:CreER; lox/lox V600E/þ specific memory. Our results also identify a novel therapeutic PTEN ; BRAF (BP) mice bred onto a C57BL/6 back- strategy of combining OX40 agonist antibodies with targeted ground were kindly provided by Dr. Bosenberg (Yale University therapy in patients with cancer, particularly those with tumors School of Medicine, New Haven, CT). Pmel-1 TCR/Thy1.1 mice with loss of the tumor suppressor PTEN. were from in-house breeding colonies. All mice were maintained in a specific pathogen-free barrier facility at MDACC. All studies were conducted in accordance with the MDACC and GlaxoS- mithKline (GSK) Policy on the Care, Welfare, and Treatment of cells and stem tumor development in patients with a variety of Laboratory Animals. All animal experiment protocols were tumors (5). However, a durable and effective antitumor immune reviewed by the Institutional Animal Care and Use Committee response only can be achieved in a small percentage of patients either at GSK or at MDACC, the institution where the work was with cancer treated with immune checkpoint blockade (ICB; performed. ref. 6). One mechanism of primary resistance to ICB is insufficient tumor-reactive T cells in patients with nonimmunogenic Caspase-3–based T-cell killing assay tumors (7). Under the notion that activation of T-cell costimu- Patient-derived tumor cells were labeled with DDAO Dye latory signaling pathways can augment the generation of effector (Thermo Fisher Scientific) according to the manufacturer's and memory T cells (8), more studies are focused on targeting instructions. Peripheral blood mononuclear cells (PBMCs) from T-cell costimulatory receptors to overcome primary resistance to healthy donors were isolated from buffy coats (Gulf Coast ICB therapy in patients with cancer. One such prominent T-cell Regional Blood Center) and irradiated with 5,000 rad of gamma costimulatory molecule is OX40. Indeed, early phase clinical radiation. Irradiated PBMCs were washed with PBS and then trials evaluating agonist antibodies targeting the OX40 pathway incubated with 10 mg/mL full length or Fc-fragment deleted alone or in combination with ICB in patients with cancer are anti-human OX40 (GSK3174998, GSK) at 37C for 1 hour. ongoing, such as NCT02221960 (formerly of MedImmune), Antibody-pulsed PBMCs were mixed with DDAO-labeled tumor NCT02528357 (GlaxoSmithKline), and NCT02554812 (Pfizer). cells and autologous TILs at 37C for an additional 3 hours. The While these trials have begun, an improved understanding of the ratio of T cells to PBMCs used in this assay was 1:1. To impact of OX40 activation on immune effector cells may help to evaluate the effect of the activation of the OX40 pathway in þ optimize the clinical evaluation of OX40-based immunotherapy murine CD8 T cells, we cross-linked anti-mouse OX40 antibody and develop novel combinatorial approaches to treat patients by pretreating bone marrow–derived dendritic cells (DCs) with cancer with primary resistance to ICB. from C57BL/6 mice with 10 mg/mL anti-murine OX40 antibody Here, by utilizing melanoma patient-derived cell lines and at 37C for 1 hour. After washing with PBS, antibody-pulsed DCs þ multiple preclinical models, we sought to determine the role of were mixed with gp100-specific CD8 Pmel-1 T cells and the OX40 pathway in regulating the effector function of tumor- MC38/gp100 tumor cells for an additional 3 hours. The ratio of reactiveT cellsandevaluatethetherapeuticpotentialof combining DCs to T cells was 1:1. The cell mixtures were then permeabilized OX40 agonist antibody with cancer-targeted therapy. Our results with Fix/Perm Solution (BD Biosciences) for 20 minutes describe the value of an OX40 agonist antibody to augment T-cell– at room temperature and stained with a PE-conjugated anti- mediated antitumor response by directly enhancing proliferation cleaved caspase-3 mAb (BD Biosciences) as described previous- þ and cytotoxicity of CD8 tumor-reactive T cells. This study also ly (11). Samples were analyzed using a FACSCanto II (BD þ provides critical rationale for the clinical evaluation of the com- Biosciences). The percentage of cleaved caspase-3 tumor cells bination of an OX40 agonist antibody and a selective PI3Kb was calculated and used to determine the extent of T-cell–induced inhibitor in patients with immunoresistant PTEN loss tumors. tumor apoptosis.

Retroviral transduction of pmel-1 T cells Material and Methods Full-length human OX40 was amplified and cloned into a Cell lines and mice retroviral vector, pMXs-IG, which was kindly provided by Dr. Human Mel2399, Mel2559, and their autologous tumor- Kitamura (University of Tokyo, Japan; ref. 12). The retroviral infiltrating lymphocytes (TILs) were established from patients vector expressing an enhanced firefly luciferase was generated in with metastatic melanoma enrolled in the adoptive T-cell therapy our previous study (13). Retroviral vectors and the packaging (ACT) trial at MD Anderson Cancer Center (MDACC, Houston, vectors were transiently cotransfected into the packaging cell line, TX) as described previously (9). The murine MC38/gp100 cell line Plate-E, using Lipofectamine 2000 (Invitrogen). Supernatants

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containing viral particles were used to infect preactivated spleno- cocktail of antibodies against intracellular markers. Stained sam- cytes from pmel-1 mice as described previously (10). Three days ples were analyzed with a FACSCanto II or a Helios Mass Cyt- after transduction, transduced pmel-1 T cells were sorted using a ometer (Fluidigm). Antibody details for the ﬂow cytometry and FACSAria (BD Biosciences) based on the expression of appropri- mass cytometry staining panels used in this study are provided in ate reporter genes embedded in the expression vectors. Supplementary Table S1.

Tumor and vaccination models Statistical analyses To determine the in vivo effect of targeting OX40 on the function Summary statistics (e.g., mean and SEM) of the data are þ of tumor-reactive CD8 T cells, luciferase-expressing pmel-1 T reported. Assessments of differences in continuous measurements cells were transferred into C57BL/6 albino mice bearing MC38/ between two groups were made using two-sample t test posterior gp100 tumor as described previously (10). One-hundred micro- to data transformation (typically logarithmic, if necessary) or grams of anti-mouse OX40 (OX-86, BioXCell) or mouse anti- Wilcoxon rank-sum test. Differences in tumor size and T-cell human OX40 (Kindly provided K.S. Voo; ref. 14) was intraper- numbers among several treatments were evaluated using ANOVA itoneally administered to tumor-bearing mice (two-times per models. The Kaplan–Meier method and log-rank test were used to week). Tumor size was monitored every two days, and in vivo compare survival between groups. P < 0.05 was considered bioluminescence imaging analyses were performed by using an statistically significant. Graph generation and statistical analyses IVIS 200 System (Xenogen) on day 6 after T-cell transfer. were performed using GraphPad Prism (version 7) and R software To evaluate the antitumor activity of anti-OX40 alone and in programming language (version 3.1.0). lox/lox combination with PI3Kb inhibition, Tyr:CreER; PTEN ; V600E/þ BRAF mice (BP mice, 6–8 weeks of age) were treated with 4-hydroxytamoxifen to induce tumor formation. Tumor-bearing Results mice were randomized into four groups to receive anti-OX40 and/ The OX40 pathway plays a critical role in regulating the or GSK2636771. GSK2636771 (GSK) was suspended in 1% (w/v) antitumor function of tumor-infiltrating T cells in patients with methylcellulose and administered to mice daily by oral gavage at a melanoma dose of 30 mg/kg. Anti-OX40 (OX-86, BioXCell) was adminis- To determine the importance of the OX40 pathway in regu- tered at a dose of 50 mg/per mouse. The relevant solvent and lating the effector function of TILs in patients with melanoma, we control rat IgG antibody (Sigma) were administered to animals in first assessed the expression of OX40 on TILs before and after the control group. restimulation with autologous tumors. We used two tumor- Because our previous study showed that CD40 agonistic anti- reactive TIL cell lines previously generated from patients with body can promote in vivo proliferation and activation of T advanced melanoma (11, 17). Cryopreserved TILs were thawed cells (15), when we evaluated the in vivo effect of anti-OX40 and cultured in fresh culture medium in the presence of IL2 for monotherapy and in combination with selective PI3Kb inhibition 3–4 days. Revived TILs were incubated with autologous tumors at on antigen-specific T cells, we redesigned our vaccine model to the varying ratios of T cells to tumor cells (E:T ratio), and the eliminate the possible confounding effects of a CD40 antibody- expression level of OX40 on the surface of TILs over time was containing immunoadjuvant on the antitumor activity of an determined by flow cytometry analysis (Fig. 1A). The majority of þ þ OX40 agonist (11, 16). Briefly, C57BL/6 mice received 1,000 resting CD4 TILs, but less than 10% of CD8 TILs, expressed þ þ na€ve pmel-1 T cells intravenously and were vaccinated with two OX40 before restimulation. The percentage of OX40 CD4 and þ distinct subcutaneous injections in each flank with 100 mLof CD8 TILs peaked at 6 hours after TCR stimulation with autol- saline containing 100 mg of human gp10025–33. In addition, ogous tumors. Seventy-2 hours after stimulation, the percentage þ vaccinated mice received 100,000 IU rhIL-2 protein i.p. once on of OX40 TILs returned to baseline levels. To validate that the the day of vaccination and twice daily on the next 2 days and were OX40 expression on restimulated TILs is tumor dependent, we þ topically treated with 50 mg of 5% imiquimod cream (Aldara, assessed the percentage of OX40 TILs after a 12-hour coincuba- Fougera) on the vaccination site once after each vaccination. tion with tumors at E:T ratios ranging from 0.1:1 to 3:1. The results showed that increasing the E:T ratio can enhance the percentage of þ þ þ Luminex assay and profiling of tumor-infiltrating immune cells both OX40 CD4 and CD8 TILs (Supplementary Fig. S1A). Serum, spleen, and tumor tissue samples were collected from These results demonstrate that OX40 expression on patient- BP mice on day 6 after treatment. Twenty-five microliters of serum derived TILs is inducible and under the control of TCR signaling. þ from each mouse was assayed using the MILLIPLEX mouse Notably, OX40 was expressed on a significant portion of CD8 cytokine/chemokine panels I, II, and III according to the manu- TILs (50%) at 6 hours after encountering autologous tumors facturer's protocol (EMD Millipore). The concentration of each (Fig. 1A), suggesting that activation of the OX40 pathway had the þ cytokine/chemokine present in the serum samples was measured potential to directly modulate the function of CD8 T cells at using a Luminex 200 system (Luminex Corporation). Fresh tumor tumor sites. To test this hypothesis, we used a cytotoxicity tissues were incubated with RPMI medium containing 1 mg/mL assay based on the expression of cleaved caspase-3 in tumor collagenase and 100 mg/mL hyaluronidase (Sigma-Aldrich) at cells to evaluate whether activation of the OX40 signaling can 37C for 60 minutes, and manually dissociated to generate alter cytotoxicity of patient-derived TILs against autologous single-cell suspensions. Single-cell suspensions from tumor or tumors. Given that immobilization of anti-OX40 antibody is spleen tissues were then washed twice with staining buffer and required to activate the OX40 pathway in T cells (14), gamma- incubated with a cocktail of antibodies targeting surface markers irradiated PBMCs were pulsed with anti-human OX40 (hOX40) at 4C for 30 minutes. Cells were then fixed and permeabilized antibody (GSK3174998) for 1 hour and used to stimulate using the Foxp3 Fix and Permeabilization Kit according to the TILs in the presence of autologous tumors. When compared manufacturer's protocol (eBioscience), and then incubated with a with Fc-fragment–deleted anti-hOX40 antibody, full-length

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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 OX40 expression on both CD4þ and CD8þ TILs upon restimulation by autologous tumor cells. Two patient-derived TIL lines, TIL2399 and TIL2559, were cultured in T-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. The percentage of TILs expressing OX40 was determined by flow cytometry analysis at indicated the timepoints. B, Cross-linked anti-OX40 antibody enhanced the cytotoxicity 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 37C for 1 hour. After washing with PBS, antibody-pulsed PBMCs were mixed with DDAO-labeled tumor cells (Mel2559) and autologous TILs (TIL2559) at 37C for an additional 3 hours. The cell mixtures were stained intracellularly with an anti-cleaved caspase-3 antibody. The cytotoxicity 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.

anti-hOX40–pulsed PBMCs significantly increased TIL-induced day 6 after T-cell transfer, the difference between these two group apoptosis of tumors (Fig. 1B). In contrast, treatment with anti- was not statistically significant (P ¼ 0.053; Fig. 2C). hOX40–pulsed PBMCs alone had no impact on tumor apoptosis. Given that an OX40 agonist antibody has been reported to þ In addition, anti-mouse OX40-pulsed DCs enhanced murine enhance the proliferation of CD4 Th cells and suppress the þ tumor apoptosis induced by tumor-reactive CD8 T cells (Supple- function of T regulatory (Treg) cells (14), the OX40 agonist– þ mentary Fig. S1B). Although OX40 was not highly expressed on enhanced antitumor activity of transferred CD8 T cells observed þ resting cytotoxic CD8 TILs, our results suggest that tumor exposure in this model may have been achieved by indirect regulation via þ þ induced the expression of OX40 on CD8 TILs and that activation CD4 T cells (18). To test whether anti-OX40 treatment can þ þ of OX40 signaling enhances the cytotoxic function of CD8 TILs. directly promote CD8 T-cell function, we modified our murine ACT model to ensure that the OX40 agonist antibody only targeted þ Activation of the OX40 pathway improves antitumor activity of OX40-expressing transferred CD8 T cells. We first transduced full- þ CD8 T cells and facilitates the generation of tumor-specificT- length human OX40 (hOX40) cDNA into murine pmel-1 T cells, þ cell memory and adoptively transferred hOX40-expressing CD8 pmel-1 T cells To characterize the in vivo effects of OX40 agonist antibody on into tumor-bearing mice. Instead of using the mOX40 agonist, we þ antitumor activity of CD8 T cells, we adoptively transferred treated all experimental mice with either isotype control antibody or þ luciferase-expressing tumor-reactive CD8 T cells (pmel-1) into anti-human OX40 antibody. Reduced tumor size was observed in tumor-bearing mice 1 day after sublethal irradiation, which is mice from the anti-hOX40 group as early as 2 days after the first dose required for expansion of transferred T cells. These mice were then of antibody treatment (Fig. 2D). Bioluminescence imaging analysis treated with either a control antibody or an anti-mouse OX40 of mice on day 6 after T-cell transfer revealed that the number of þ (mOX40) antibody as shown in Fig. 2A. The gp100-expressing transferred CD8 T cells in the tumors was comparable between the MC38 tumors in mice treated with pmel-1 T cells grew signifi- control and anti-hOX40 groups (Fig. 2E). Although OX40 agonist cantly slower than those not treated with T cells (P < antibody treatment had limited impact on directly regulating tumor þ 0.0001; Fig. 2B). Importantly, anti-mOX40 treatment significant- trafficking of CD8 T cells, data from both models supports that ly delayed tumor growth in all T-cell–treated mice (P < activation of OX40 signaling promotes the effector function of þ 0.001; Fig. 2B). We also used bioluminescence imaging analysis tumor-reactive CD8 T cells. to determine the change in tumor trafficking of transferred tumor- We then examined the impact of anti-OX40 on the generation þ reactive T cells in response to anti-mOX40 treatment. Although of antigen-specific CD8 memory T cells using a murine vaccine the average bioluminescence intensity at the tumor site in mice model (Fig. 3A; refs. 11, 16). The composition of adjuvants in our treated with anti-mOX40 was higher than the control group on previously described murine vaccination model was simplified to

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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 to evaluate 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 of anti-mouse OX40 antibody. Pmel-1 T cells that express a TCR specifically recognizing a melanoma tumor antigen (gp100) were modified to express luciferase for in vivo monitoring of tumor trafficking. Luciferase-expressing pmel-1 T cells were transferred into mice bearing gp100-expressing MC38 tumors (N ¼ 4 per group). All experimental mice were then treated with DC vaccine and IL2 as described previously (41). One-hundred micrograms per dose of anti-OX40 or control 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 mice with 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 did not significantly alter tumor tracking of transferred T cells. D, Anti-human OX40 antibody facilitated human OX40-expressing pmel-1 T cells control of the growth of MC38/gp100 tumors (N ¼ 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 after two-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. avoid confounding effects of the anti-CD40 antibody on anti- successfully suppressed the development of gp100-expressing OX40 antibody activity. Briefly, fresh splenocytes from pmel-1 tumors, indicating that OX40 agonists can induce the generation mice were adoptively transferred into C57BL/6 mice. Experimen- of tumor-reactive memory T cells (Fig. 3C). tal mice were then vaccinated with the gp100 peptide on day 0 and day 28, and treated with anti-mOX40 on days 0, 4, 7, 12, 28, 31, OX40 agonist antibody synergizes with GSK2636771 in 35, and 40. One week after the last anti-OX40 treatment, exper- controlling the development of PTEN-null melanoma imental mice were challenged with gp100-expressing MC38 Previously, we demonstrated that oncogenic activation of the tumor cells. By monitoring the percentage of transferred pmel- PI3K pathway by PTEN loss promotes tumor-associated immu- 1 T cells in PBMCs, we found that anti-OX40 enhanced the nosuppressive mechanisms and is associated with poor clinical proliferation of pmel-1 T cells after initial antigen stimulation outcomes in patients with melanoma treated with anti–PD-1 (on day 5), and this positive effect of anti-OX40 was dose (11). We also found that Braf-mutant, PTEN-null melanomas V600E/þ lox/lox dependent (Fig. 3B). Five days after the booster vaccine (on day developed in Tyr:CreER; BRAF ; PTEN mice (BP mice) 33), the percentage of antigen-specific T cells in peripheral blood are resistant to immune checkpoint inhibitory antibodies. How- þ CD8 T cells in mice was also significantly higher than the rest of ever, improved tumor growth inhibition was observed with the vaccinated mice (Fig. 3B). Moreover, administration of 200 mgof combination of anti–PD-1 antibodies with GSK2636771, a anti-OX40 to gp100-vaccinated mice before tumor inoculation PI3Kb-selective inhibitor, which was selected on the basis of data

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Figure 3. In vivo OX40 agonist antibody treatment enhanced the proliferation of tumor-reactive T cells upon TCR stimulation and induced the generation of tumor-specific T-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-reactive T cells. C57BL/6 mice were transferred with the splenocytes from pmel-1 mice and vaccinated with gp100 peptide. Vaccinated mice received either control antibody or anti-mouse OX40 antibody. After 4 weeks, mice received a second gp100 peptide vaccine (booster). Gp100-expressing MC38 tumors were subcutaneously 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 with OX40 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 blood after antigen stimulation. The pairwise multiple comparisons after two-way ANOVA test demonstrated statistical significance (, P < 0.05): control/a-OX40 50 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/gp100 tumors in vaccinated mice treated with anti-OX40 (N ¼ 8 per group). Representative data from two independent experiments are shown.

supporting a role for this PI3K isoform–selective inhibition in bined anti-OX40 with GSK2636771 in the BP model (Fig. 4A). BP cells with loss of PTEN (11, 19, 20). The combination of pem- mice bearing measurable melanoma lesions were randomized brolizumab and GSK2636771 is now being evaluated in a phase and treated with isotype control antibody, GSK2636771, anti- I/II clinical trial (NCT03131908). OX40 antibody, or a combination of the two agents. Single-agent To evaluate the effectiveness of combining T-cell costimulatory anti-OX40 and single-agent GSK2636771 both failed to signiﬁ- receptor-based immunotherapy and targeted therapy, we com- cantly inhibit tumor growth, but the combination was highly

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 the lox/lox V600E/þ PI3Kb inhibitor (GSK2636771) is shown. Melanoma was induced in a group of Tyr:CreER; PTEN ; BRAF mice. Mice with measureable tumors were randomized and treated with control, GSK2636771 (30 mg/kg/day), anti-mouse OX40 (50 mg/dose), and the combination of both reagents. B, Tumor size was monitored in each of the treatment groups every 2 days. The pairwise multiple comparisons after two-way ANOVA test were used to determine statistical signiﬁcance. , P < 0.05. C, Kaplan–Meier survival curves of mice treated with GSK2636771 and/or anti-mouse OX40. Log-rank test demonstrated statistical signiﬁcance (P < 0.05): GSK2636771þanti-OX40 versus control, GSK2636771, and anti-OX40 (N ¼ 4–7). Data presented are a summary of two independent experiments.

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Figure 5. OX40 agonist antibody in combination with PI3Kb-selective inhibition altered the immune cell proﬁle and the serum concentration of cytokine/chemokines produced by T cells. Tyr:CreER; PTENlox/lox; BRAFV600E/þ mice with measureable tumors were treated with control, GSK2636771, anti-mouse OX40, and the combination 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 different treatment groups. A, The serum levels of T-cell–associated cytokines/chemokines. Serum from each experimental mouse was collected and used to measure the concentration of 43 cytokines/chemokines using the MILLIPLEX MAP mouse cytokine/chemokine panels. The average of the serum cytokine/chemokine concentration in each group is shown. , P < 0.05; , P < 0.01; , P < 0.0001 (N ¼ 3). B, The results of CyTOF analysis revealed the systemic effects of anti- OX40 alone or in combination with PI3Kbi. Spleens were collected from mice in the different treatment groups and processed into single-cell suspensions at the concentration of 20 million cells/mL. Equal amounts of single-cell suspensions from experimental mice in each group were pooled (N ¼ 3). Pooled samples of control, 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 ratio of 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 a summary of two independent experiments.

effective and markedly extended the survival of BP tumor-bearing Combining anti-OX40 with a PI3Kb inhibitor enhances T-cell– mice (the median survival times of control, GSK2636771, anti- mediated antitumor immune activity OX40, and combination groups are 14.5, 18, 14, and 30 days, We then explored the underlying mechanisms by which PI3Kb respectively; P ¼ 0.0021; Fig. 4B and C). A linear mixed mod- inhibition synergizes with anti-OX40 to control Braf-mutant, el (21) determined that the antitumor effect of GSK2636771 with PTEN-null melanomas. Additional tumor-bearing BP mice were anti-OX40 was synergistic (P ¼ 0.0004; Supplementary Fig. S2). treated with anti-OX40 and/or GSK2636771 as described above. Importantly, no overt adverse effects or toxicities were observed On day 6, serum, spleen, and tumor tissue samples were collected with the combination treatment. We further tested whether for immune profiling. We measured the serum concentrations of a GSK2636771, anti-OX40, or the combination affected the pro- broad set of chemokines/cytokines in each experimental mouse liferation of antigen-specific T cells upon in vivo antigen stimu- using Luminex assays. Among 43 tested chemokines/cytokines, lation. These experiments showed that the combination treat- the combination treatment significantly increased the serum ment did not significantly reduce whole-blood cell counts or concentrations of CCL2, CCL4, CCL15, CXCL10, and G-CSF in inhibit the proliferation of gp100-specific T cells upon gp100 comparison with the monotherapy or control treatments (Fig. 5A; peptide immunization (Supplementary Fig. S3). Taken together, Supplementary Fig. S4). Two of these factors, CCL4 and CXCL10, these data suggest that combining anti-OX40 with GSK2636771 are mainly produced by memory and/or effector T cells. In is another potentially effective strategy to overcome immune addition, the serum levels of IFNg, another important antitumor resistance in melanomas with PTEN loss. cytokine produced by T cells, were significantly higher in mice

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Figure 6. þ OX40 agonist antibody in combination with PI3Kb-selective inhibition promoted the proliferation of tumor-infiltrating CD8 T cells in mice with PTEN-loss tumor. Tyr:CreER; PTENlox/lox; BRAFV600E/þ mice with measureable tumors were treated with control, GSK2636771, anti-mouse OX40, and the combination of both reagents (N ¼ 3). On day 6 after treatment, tumor tissues were harvested, weighted, and stained with antibodies for flow cytometry analysis. A, The total number of CD4þ and CD8þ T cells in tumors from mice treated with anti-OX40 and/or GSK2636771. B, The percentage of Treg cells (CD25þFOXp3þ) in CD4þ T cells in tumors from mice treated with anti-OX40 and/or GSK2636771. C, The percentage of Ki-67þ CD4þ and CD8þ T cells in tumors from mice 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 of two independent experiments.

who received the combination treatment than in mice treated Discussion with the control antibody or PI3Kb inhibitor alone (Fig. 5A). In this article, we examined the expression of OX40 on TILs Next, we characterized the function and phenotype of immune derived from patients with melanoma and tested whether stim- cells in spleens from mice in the different treatment groups using a ulating OX40 signaling can promote cytotoxicity of TILs against 24-channel mass cytometry (CyTOF) panel (Supplementary autologous tumor cells. Data from preclinical tumor models Table S1). High-dimensional analysis using SPADE was per- confirmed that OX40 agonist antibody can improve T-cell– formed to examine the changes in immune cells in the treatment mediated antitumor immune responses by OX40 receptor groups. Anti-OX40 monotherapy reduced the percentage of M2 þ engagement on CD8 T cells and inducing a protective tumor- macrophages, which are immunosuppressive immune cells specific T-cell memory. To develop effective therapeutic found in peripheral lymphoid organs. In comparison with approaches in patients with cancer who fail to respond to ICB, anti-OX40 monotherapy, combination treatment further reduced we used a transgenic Braf-mutant and PTEN-loss murine model, the percentage of M2 macrophages and significantly increased the which can spontaneously develop immune-resistant melanomas, expression of Ki-67 in T cells, DCs, and M1 macrophages, suggest- to evaluate the efficacy of combining OX40 agonist antibody with ing enhanced proliferation of antitumor immune cells (Fig. 5B). targeted therapy. The combination of an OX40 agonist antibody Because of poor tumor infiltration of immune cells in this tumor and a selective PI3Kb inhibitor successfully potentiated the pro- model, the number of immune cells at the tumor site was liferation of antitumor immune cells and suppressed tumor insufficient to perform CyTOF analysis. Therefore, we evaluated development in mice bearing Braf-mutant and PTEN-loss the changes of T-cell compartments at the tumor sites by a five- melanoma. channel flow cytometry panel (Supplementary Table S1). OX40, also known as TNFRSF4 or CD134, belongs to the TNF Although GSK2636771 significantly increased the number of þ receptor superfamily (22). The engagement of three molecules of CD8 T cells within tumors, there was no significant difference þ OX40 and trimeric OX40 ligand (OX40L) initiates the signaling in the number of tumor-infiltrating CD8 T cells in the cascade through TNF receptor–associated factors and eventually GSK2636771 monotherapy group versus the combination treat- drives NF-kB activation (8). Although OX40 expression can be ment group (Fig. 6A). In addition, neither monotherapy treat- þ þ induced by TCR activation in both CD4 and CD8 T cells, the ment nor the combination significantly altered the total number þ þ expression of OX40 in CD4 TILs is significantly higher than in of tumor-infiltrating CD4 T cells (Fig. 6A). In addition, the þ CD8 TILs (23, 24). In addition, in vitro and in vivo studies using number of Tregs in the tumors from the combination treatment viral infection and autoimmune disease models have demon- group was comparable with those of the monotherapy-treated þ strated that the effect of OX40 activation on CD8 T cells is largely tumors (Fig. 6B). By using the expression of Ki-67 to determine þ indirect and is mediated by OX40 regulation of CD4 Th cell T-cell function at tumor sites, we observed that a significant þ þ function (25–27). The activation of the OX40 pathway in regu- increase in the percentage of Ki-67 CD8 T cells, but not in the þ þ þ latory CD4 T (Treg) cells has also been reported to blunt the percentage of Ki-67 CD4 T cells, was detected with the com- immunosuppressive function of Treg cells (14). Therefore, the bination versus each of the other treatment groups (Fig. 6C). current working model of OX40 agonist antibody function in These results suggest that combining GSK2636771 with anti- þ tumors mainly focuses on its effect on CD4 T cells. In our studies, OX40 promotes T-cell–mediated antitumor immune responses þ we evaluated the OX40 expression levels in established TIL lines by inducing robust proliferation of CD8 tumor-infiltrating from patients with advanced melanoma under different culture T cells.

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OX40 Agonist–Based Cancer Immunotherapy

conditions. Consistent with the results from other types of cancer, reagents, such as TLR9 agonist in a spontaneous breast cancer þ OX40 is predominantly expressed on resting CD4 TILs. How- murine model (35). However, when evaluating the combination ever, upon encountering autologous tumor cells, the OX40 of anti-OX40 and anti–PD-1, two research groups independently þ expression on CD8 TILs is significantly upregulated and is demonstrated that concurrent treatment using these two agents restored to the baseline 72 hours after TCR stimulation. induced T-cell apoptosis and produced antagonistic antitumor These results prompted us to test whether OX40 agonist antibody responses. Enhanced antitumor effect was only observed in mice þ can directly potentiate the function of tumor-reactive CD8 TILs. sequentially treated with anti-OX40 and anti–PD-1 (36, 37). A This hypothesis was first supported by data demonstrating that similar potentially antagonistic antitumor effect has been in vitro, anti-OX40 antibody can promote the proliferation of observed with the combination of immunotherapy and targeted þ na€ve CD8 T cells after anti-CD3 stimulation (14). In our study, therapy. In that, although both CpG-based tumor vaccine and by using patient-derived TILs and paired autologous tumor cells, BRAF inhibitor have therapeutic benefit as monotherapies in we found that cross-linked OX40 agonist antibody facilitated patients with cancer, combining CpG with BRAF inhibitor negates tumor apoptosis induced by autologous TILs in vitro. Given that the antitumor effect of BRAF inhibitor in Braf-mutant tumors in a the tumor-specific cytotoxicity of TILs used in this study has been B-cell–dependent manner (38). Therefore, to develop potent previously shown to be largely dependent on the expression of therapeutic strategies for OX40-based cancer treatment, we need MHC class I molecules (11), our results suggest that OX40 to not only rationally choose combination partners with com- signaling can directly regulate cytotoxicity of tumor-reactive plementing effects, but also optimize treatment schedules to þ þ CD8 TILs. In addition, we examined the changes in CD8 maximize the antitumor effect of anti-OX40. T cells in response to anti-OX40 treatment using multiple murine Melanomas that spontaneously develop in transgenic mice tumor models. Our in vivo data further demonstrated that anti- bearing the Braf V600E mutation and PTEN loss in melanocytes, OX40 treatment enhances antitumor activity of tumor-reactive display primary resistance to cancer immunotherapy, due to lack þ CD8 T cells. Furthermore, we used a vaccination model estab- of tumor-associated antigens and upregulated immunosuppres- lished in our previous studies to evaluate T-cell memory forma- sive factors induced by oncogenic activation of the PI3K path- þ tion (16) and found that anti-OX40 treatment promoted CD8 way (11, 39, 40). In this study, we used this immune-resistant T-cell–mediated antitumor memory induced by antigen vaccina- tumor model to evaluate the therapeutic efficacy of anti-OX40 in tion. In particular, when treating tumor-bearing mice with anti- combination with PI3K inhibition. Although anti-OX40 mono- human OX40 antibody without cross-reactivity to mouse OX40, therapy did not effectively control tumor development, concur- we consistently observed improved tumor suppression by human rent treatment of anti-OX40 and PI3Kb-selective inhibition sig- þ OX40–expressing CD8 T cells. These results imply that the direct nificantly delayed tumor growth and extended the survival of þ role of OX40 signaling in tumor-reactive CD8 T cells should not mice bearing Braf-mutant and PTEN-loss melanomas. Unlike the be overlooked. combination of PI3Kb inhibition and ICB, this combinatorial þ In clinic, the potential of OX40 as a target for cancer immu- approach did not significantly increase the number of CD4 and þ notherapy was initially tested by using a murine anti-human CD8 T cells at the tumor sites but promoted the proliferation of þ OX40 IgG1 mAb, 9B12 (28). Although no patients achieved a CD8 T cells at the tumor sites. Anti-OX40 plus PI3Kb inhibitor clinical response based on the RECIST, 12 of 30 treated patients treatment also systemically enhanced the proliferation of antitu- had at least one regressed metastatic nodule. Multiple fully mor immune cells but reduced the number of immunosuppres- human or humanized OX40 agonist antibodies have been gen- sive M2 macrophages. We also found elevated serum levels of erated in the last 2 decades. At least five different antibodies have cytokine/chemokines, which were predominantly produced by entered clinical development, including GSK3174998 (GSK), effector T cells in mice treated with the combination therapy. In INCAGN01949 (Agenus), MEDI0562 (formerly of MedIm- addition, our studies showed that this combination did not mune), MOXR0916 (Genentech), and PF-04518600 (Pfizer; increase the susceptibility of T cells to activation-induced apo- refs. 29–33). Similar to the preclinical results from murine tumor ptosis. Overall, our results offer the first preclinical evidence models (34), the early data from two phase I clinical trials showed demonstrating that combining anti-OX40 with PI3Kb inhibitor anti-OX40 monotherapy was well tolerated in patients with could be an effective treatment for patients with PTEN-loss cancer, with only one serious treatment-related grade 3 adverse tumors. These results also provide the rationale to clinically test event (pneumonitis responsive to corticosteroids) of 71 patients this combination in patients with immunoresistant PTEN-loss reported (29, 30). These results suggest that OX40 agonist anti- tumors. body treatment in patients with cancer is generally well-tolerated. Taken together, our studies suggest that OX40 agonist–based In addition, up to 200 mg of anti-OX40 per dose for 2 weeks had combination treatment can induce a robust and durable antitu- no adverse effect on the health and well-being of experimental mor immune response by promoting effector T-cell function and mice in this study. the generation of memory T cells. The antitumor effect of OX40 agonist antibody in patients with cancer has not been fully elucidated. However, primary and Disclosure of Potential Conflicts of Interest acquired resistance to anti-OX40 monotherapy are expected in W. Peng reports receiving speakers bureau honoraria from Bristol-Myers patients with cancer due to a wide variety of tumor-associated Squibb and reports receiving commercial research grants from immunosuppressive factors. Therefore, combining anti-OX40 GlaxoSmithKline. H. Jackson is an employee of and has ownership interests therapy with other treatments targeting these tumor-associated (including patents) at GlaxoSmithKline. K.S. Voo is listed as a co-inventor on a patent on Anti-OX40 antibody for the treatment of cancer which is immunosuppressive factors may result in better response rates owned by MD Anderson Cancer Center and licensed to GlaxoSmithKline. and improved overall survival in patients with cancer. In addition, H.A. Tawbi is a consultant/advisory board member for Bristol-Myers eradication of well-established tumors has been reported in mice Squibb, Novartis, Merck, Genentech and Array, and reports receiving treated with anti-OX40 in combination with other immune commercial research grants from Bristol-Myers Squibb, Merck, Genentech,

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GlaxoSmithKline and Celgene. M.A. Davies is a consultant/advisory board Administrative, technical, or material support (i.e., reporting or organizing member for Novartis, GlaxoSmithKline, Roche Genentech, Bristol-Myers data, constructing databases): W. Peng, L.J. Williams, N. Yanamandra, P. Hwu Squibb, Sanofi Aventis, NanoString, and reports receiving commercial Study supervision: W. Peng, G. Lizee, J. Smothers, R. Srinivasan research grants from GlaxoSmithKline, Bristol-Myers Squibb, Roche/ Genentech, Sanofi Aventis, AstraZeneca and Oncothyreon. A. Hoos is an Acknowledgments employee of and has ownership interests (including patents) at This work was supported in part by the following NCI grants: R01CA187076 GlaxoSmithKline and Imugene, and is a consultant/advisory board member (to P. Hwu and M.A. Davies), P50CA093459 (to UT M.D. Anderson Cancer for CRI. R. Srinivasan has ownership interests (including patents) at Center SPORE in Melanoma), T32CA009666-21(to M.A. Davies), and GlaxoSmithKline. E.M. Paul is an employee of and has ownership interests P30CA016672 (to UT MDACC CCSG for the Flow Cytometry & Cellular (including patents) at GlaxoSmithKline. N. Yanamandra is an employee Imaging facility), by philanthropic contributions to MDACC Melanoma Moon of and has ownership interests (including patents) at GlaxoSmithKline. Shots Program; Melanoma Research Alliance Young Investigator Award (to fl P. Hwu has ownership interests (including patents) at Dragon yand W. Peng, 558998); Dr. Miriam and Sheldon G. Adelson Medical Research fl Immatics, is a consultant/advisory board member for Dragon y, Immatics, Foundation; Aim at Melanoma Foundation, Miriam and Jim Mulva Research fi fl GlaxoSmithKline and Sano .Nopotentialconicts of interest were Fund; and by Cancer Prevention and Research Institute of Texas (to P. Hwu disclosed by the other authors. RP170401 and to J.A. McKenzie RP140106 and RP170067). The authors would like to thank the past and present TIL lab members at Authors' Contributions MDACC: Orenthial J. Fulbright, Arely Wahl, Esteban Flores, Shawne T. Thorsen, Conception and design: W. Peng, K.S. Voo, J. Smothers, E.M. Paul, Rene J. Tavera, Renjith Ramachandran, Audrey M. Gonzalez, Christopher N. Yanamandra, P. Hwu Toth, Seth Wardell, and Rahmatu Mansaray as well as Drs. Chantale Bernatchez, Development of methodology: W. Peng, K.S. Voo, P. Hwu Cara Haymaker, Marie-Andree Forget, and Shruti Malu for generation of Acquisition of data (provided animals, acquired and managed patients, research TIL and tumor lines. 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. Leahey The costs of publication of this article were defrayed in part by the payment Analysis and interpretation of data (e.g., statistical analysis, biostatistics, of page charges. This article must therefore be hereby marked advertisement computational analysis): W. Peng, C. Xu, B. Melendez, H. Jackson, K.S. Voo, in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. J. Wang, H.A. Tawbi, M.A. Davies, A. Hoos, N. Yanamandra, P. Hwu Writing, review, and/or revision of the manuscript: W. Peng, J.A. McKenzie, Received April 21, 2019; revised June 10, 2019; accepted July 26, 2019; K.S. Voo, J. Wang, G. Lizee, H.A. Tawbi, M.A. Davies, A. Hoos, E.M. Paul, P. Hwu published first August 1, 2019.

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Anti-OX40 Antibody Directly Enhances The Function of Tumor-Reactive CD8 + T Cells and Synergizes with PI3Kβ Inhibition in PTEN Loss Melanoma

Weiyi Peng, Leila J. Williams, Chunyu Xu, et al.

Clin Cancer Res Published OnlineFirst August 1, 2019.

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