Abstract Adoptive T cell therapy (ACT) using tumor infiltrating lymphocytes (TILs) has demonstrated efficacy in metastatic melanoma patients, with ~50% objective responses. Amongst parameters recognized as important for the efficacy of ACT is a high proportion of CD8+ T cells in the infusion product. OX40 (CD134) belongs to the tumor necrosis factor receptor super family and is mainly expressed by activated T lymphocytes. Activation of OX40 signaling promotes proliferation and survival of T-cells via the NF-kB pathway. Agonistic anti-OX40 antibodies have been developed as immunotherapy drugs for the treatment of cancer, one of which was recently shown to increase reactivity of tumor antigen-specific CD8+ T cells in patient's peripheral blood. In this work, we studied the expression of the OX40 receptor on TILs and investigated the impact of an anti-OX40 agonistic antibody on the ex vivo expansion and effector function of TILs. We found that OX40 was enriched in the CD4+ T cell subset of TILs. Its expression was up-regulated in response to anti-CD3 stimulation during the rapid expansion protocol (REP) of TIL production. An anti-OX40 monoclonal antibody was obtained from commercial sources and characterized in vitro. It was shown to bind OX40 specifically and to mediate NF-κB activation in a dose-dependent manner upon clustering. Consistent with its agonistic activity in the reporter assay, the anti-OX40 antibody also heightened IFN-γ production of OKT3-stimulated TILs, suggesting improved T cell effector function (N=6; p=0.03) and supporting its use during TIL expansion. When TILs were propagated by REP in the presence of anti-OX40, the frequency of CD8+ T cells increased significantly in the final product relative to control (N=21, p=0.001). The effect was dose-dependent. Interestingly, cell surface expression of the OX40 receptor was specifically down-regulated in the responding anti-OX40-treated TILs, suggesting a link between regulation of the OX40 signaling axis and the antibody-mediated effect on the TIL subsets. The TCR Vβ repertoire of TILs expanded with anti-OX40 was not changed significantly (N=3). However, expression of T cell surface markers was altered by the antibody, which skewed the T cell phenotype towards a differentiated and activated state. Taken together, maintenance of T cell diversity and differentiation state of the T cells suggest that anti-OX40 acted by selectively promoting the proliferation of CD8+ T cells. When assessed functionally in an IFNγ assay, the potency of the TILs produced in the presence of anti-OX40 was shown to be comparable to that of control TILs. In conclusion, our work shows that activation of OX40 signaling using an agonistic antibody could significantly improve expansion of CD8+ TILs. Implications on in vivo anti-tumor activity remain to be assessed. Citation Format: Krit Ritthipichai, Marcus Machin, Maria Fardis, Cecile Chartier. Anti-OX40 agonistic antibody enhances ex vivo CD8+ TIL expansion with increased T-cell effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-110.