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The cytokine changes in mice receiving PI3K inhibition in combination with anti-OX40 treatment.

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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 PI3Kβ 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 PI3Kβ-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 IFNγ, which are mainly produced by memory and/or effector T cells.Conclusions: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.

Current cancer vaccines induce tumor-specific T cell responses without sustained tumor regression because immunosuppressive elements within the tumor induce exhaustion of effector T cells and infiltration of immune-suppressive regulatory T cells (Tregs). Therefore, much effort has been made to generate agonistic Abs targeting members of the TNFR superfamily, such as OX40, 4-1BB, and GITR, expressed on effector T cells and Tregs, to reinvigorate T cell effector function and block Treg-suppressive function. In this article, we describe the development of a panel of anti-human OX40 agonistic mouse mAbs that could promote effector CD4+ and CD8+ T cell proliferation, inhibit the induction of CD4+ IL-10 -producing type 1 regulatory T cells, inhibit the expansion of ICOS+IL-10+ Tregs, inhibit TGF-β–induced FOXP3 expression on naive CD4+ T cells, and block natural Treg–suppressive function. We humanized two anti–human OX40 mAb clones, and they retained the potency of their parental clones. These Abs should provide broad opportunities for potential combination therapy to treat a wide realm of cancers and preventative vaccines against infectious diseases.

IL-17-producing CD4+ T cells (Th17) have recently been defined as a unique subset of pro-inflammatory helper cells whose development depends on signaling initiated by IL-6 and TGF-β, autocrine activity of IL-21, activation of STAT3, and induction of the orphan nuclear receptor RORγt. The maintenance, expansion and further differentiation of the committed Th17 cells depends on IL-1β and IL-23. IL-17 was originally found produced by human CD45RO+ memory T cells. We report that human peripheral blood and lymphoid tissue contain a significant number of CD4+FOXP3+ T cells that express CCR6 and produce IL-17 upon activation. These cells co-express FOXP3 and RORγt transcription factors and strongly inhibit the proliferation of CD4+ responder T cells. CD4+CD25high-derived T cell clones express FOXP3, RORγt and IL-17, and maintain their suppressive function. We further show that human CD4+FOXP3+CCR6- Treg cells differentiate into IL-17 producer cells upon TCR stimulation in the presence of IL-1β, IL-2, IL-21, IL-23 and human serum. This, together with the finding that human thymus does not contain IL-17-producing Treg cells, suggests that the IL-17+FOXP3+ Treg cells are generated in the periphery. IL-17-producing Treg cells may play critical roles in anti-microbial defense while controlling autoimmunity and inflammation. This work was supported by KECK Foundation 01, PP-4 and National Institute of Health Grant AI091130 (to Y.-J.L.)