Author Manuscript Published OnlineFirst on August 1, 2019; DOI: 10.1158/1078-0432.CCR-19-1259 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Anti-OX40 antibody directly enhances the function of tumor-reactive CD8+ T cells and synergizes with PI3Kβ inhibition in PTEN loss melanoma Weiyi Peng1,5*, Leila J. Williams1, Chunyu Xu1,5, Brenda Melendez1, Jodi A. McKenzie1,6, Yuan Chen1, Heather Jackson2, Kui S. Voo3, Rina M. Mbofung1,7,, Sara E. Leahey1, Jian Wang4, Greg Lizee1, Hussein A. Tawbi1, Michael A. Davies1, Axel Hoos2, James Smothers2, Roopa Srinivasan2, Elaine Paul2, Niranjan Yanamandra2* and Patrick Hwu1* 1Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX. 2Oncology R&D, Immuno-Oncology and Combinations RU, GlaxoSmithKline, 1250 S. Collegeville Rd, Collegeville, PA 19426, United States 3Oncology Research for Biologics and Immunotherapy Translation Platform, The University of Texas MD Anderson Cancer Center, Houston, TX. 4Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX. 5Present address: Department of Biology and Biochemistry, University of Houston, Houston, TX. 6Present address: Eisai Inc., Woodcliff Lake, NJ. 7Present address: Merck Research Laboratories, Palo Alto, CA. Running Title: OX40 agonist-based cancer immunotherapy Keywords: OX40, PI3K, cancer immunotherapy Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 1, 2019; DOI: 10.1158/1078-0432.CCR-19-1259 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 2 *Corresponding Authors: Patrick Hwu, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-563-1728; Fax: 713-745-1046; Email: [email protected] Niranjan Yanamandra, Immuno-Oncology and Combinations RU, GlaxoSmithKline, 1250 s. Collegeville Rd, Collegeville PA, 19426, Phone: 610-917-5123; Email: [email protected] Weiyi Peng, The University of Houston, 3517 Cullen Blvd, Houston, TX, 77204. Phone: 713-743-6941; Fax: 713-743-3415; Email: [email protected] Conflict of Interest: The authors of this publication have research support from GlaxoSmithKline (GSK). The terms of this agreement have been reviewed and approved by the University of Texas MD Anderson Cancer Center (MDACC) in accordance with its policy on objectivity in research. W. Peng received honoraria and travel support from Bristol-Myers Squibb (BMS). H. Jackson, A. Hoos, J. Smothers, R. Srinivasan, E. Paul and N. Yanamandra are full-time employees of GSK. P. Hwu is a consultant/an advisory board member for Immatics, Dragonfly, Sanofi, and GSK. M.A. Davies is an advisory board member for BMS, GSK, Novartis, Roche/Genentech, Array, Sanofi, and Vaccinex. M.A. Davies is also the PI of research funding to MDACC from GSK, AstraZeneca, Roche/Genentech, Myriad, Oncothyreon, and Sanofi-Aventis. No potential conflicts of interest were disclosed by other authors. Grant Support Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 1, 2019; DOI: 10.1158/1078-0432.CCR-19-1259 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 3 This work was supported in part by the following National Cancer Institute grants: R01CA187076 (PH&MD), P50CA093459 (UT M.D. Anderson Cancer Center SPORE in Melanoma), T32CA009666-21(MD) and P30CA016672 (UT MDACC CCSG for the Flow Cytometry & Cellular Imaging facility), by philanthropic contributions to MDACC Melanoma Moon Shots Program; Melanoma Research Alliance Young Investigator Award (WP, 558998); Dr. Miriam and Sheldon G. Adelson Medical Research Foundation; Aim at Melanoma Foundation, Miriam and Jim Mulva Research Fund; and by Cancer Prevention and Research Institute of Texas (PH, RP170401; JAM, RP140106 and RP170067). Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 1, 2019; DOI: 10.1158/1078-0432.CCR-19-1259 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 4 ABSTRACT Purpose: OX40 agonist-based combinations are emerging as a novel avenue to improve the effectiveness of cancer immunotherapy. To better guide its clinical development, we characterized the role of the OX40 pathway in tumor-reactive immune cells. We also evaluated combining OX40 agonists with targeted therapy to combat resistance to cancer immunotherapy. Experimental Design: We utilized patient-derived tumor infiltrating lymphocytes (TILs) and multiple preclinical models to determine the direct effect of anti-OX40 agonistic antibodies on tumor-reactive CD8+ T cells. We also evaluated the antitumor activity of an anti-OX40 antibody plus PI3K inhibition in a transgenic murine melanoma model (Braf-mutant, PTEN null), which spontaneously develops immunotherapy-resistant melanomas. Results: We observed elevated expression of OX40 in tumor reactive CD8+ TILs upon encountering tumors; activation of OX40 signaling enhanced their cytotoxic function. OX40 agonist antibody improved the antitumor activity of CD8+ T cells and the generation of tumor-specific T cell memory in vivo. Furthermore, combining anti-OX40 with GSK2636771, a PI3K selective inhibitor, delayed tumor growth and extended the survival of mice with PTEN-null melanomas. This combination treatment did not increase the number of TILs, but it instead significantly enhanced proliferation of CD8+ TILs and elevated the serum levels of CCL4, CXCL10, and IFN-γ, which are mainly produced by memory and/or effector T cells. Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 1, 2019; DOI: 10.1158/1078-0432.CCR-19-1259 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 5 Conclusion: These results highlight a critical role of OX40 activation in potentiating the effector function of tumor-reactive CD8+ T cells and suggest further evaluation of OX40 agonist-based combinations in patients with immune-resistant tumors. Translational Relevance: Cancer immunotherapy is revolutionizing cancer treatment. However, most patients still fail to respond to currently available immunomodulatory agents. Thus, there remains a critical need to identify novel immunoregulatory targets and rational combinatorial strategies to induce robust and durable antitumor immune responses. Here, we used patient samples and clinically relevant animal models to evaluate the immunological and antitumor effects of OX40 agonist-based immunotherapy. Our results add to the growing body of evidence that OX40 agonists can boost antitumor immune responses by modulating T cell effector function and tumor-specific memory. Our results also identify a novel therapeutic strategy of combining OX40 agonist antibodies with targeted therapy in cancer patients, particularly those with tumors with loss of the tumor suppressor PTEN. Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 1, 2019; DOI: 10.1158/1078-0432.CCR-19-1259 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 6 INTRODUCTION Several immunomodulatory agents that target T cell co-inhibitory receptors, such as PD-1 and CTLA-4, have been developed to boost T cell-mediated antitumor immune responses in cancer patients. These immunotherapies have demonstrated durable clinical benefit in many types of cancer, and immune checkpoint blockade has become a standard front-line treatment in multiple solid cancers, including melanoma, lung cancer, bladder cancer, and kidney cancer (1,2). This new clinical paradigm has shifted research efforts in tumor immunology to prioritize the identification of additional immunoregulatory targets and rational combinatorial treatments to further increase the rate of potent and durable antitumor immune responses. T cell activation is tightly regulated by two sets of signals via T cell receptors (TCR) and T cell co-signaling receptors. Positive (co-stimulatory) and negative (co- inhibitory) signals from T cell co-signaling receptors direct T cell function in response to TCR stimulation. Several studies have demonstrated that activating T cell co-stimulatory receptors, such as OX40 and 4-1BB, can facilitate T cell-mediated antitumor immunity (3,4). Moreover, disrupting T cell co-inhibitory signaling pathways, such as PD-1 and CTLA-4, has been reported to reinvigorate tumor-reactive T cells and stem tumor development in patients with a variety of tumors (5). However, a durable and effective antitumor immune response only can be achieved in a small percentage of cancer patients treated with immune checkpoint blockade (ICB) (6). One mechanism of primary resistance to ICB is insufficient tumor-reactive T cells in patients with non-immunogenic tumors (7). Under the notion that activation of T cell co-stimulatory signaling pathways can augment the generation of effector and memory T cells (8), more studies are focused Downloaded from clincancerres.aacrjournals.org