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 Ó2019 American Association for Cancer Research. and CTLA-4, has been reported to reinvigorate tumor-reactive T www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst August 1, 2019; DOI: 10.1158/1078-0432.CCR-19-1259 Peng et al. 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