Abstract 1536: Enhancing CAR T cell activity by linking IL-12 expression to the endogenous <i>PDCD1</i> promoter
Authors: Baeckseung Lee, Wai-Hang Leung, Jasdeep Mann, Kyle Havens, Joel Gay, Richard A. Morgan, Alexander Astrakhan
Journal: Cancer Research (2018)

Abstract Chimeric antigen receptor (CAR) T cells have shown great promise in treating certain late stage hematological malignancies. While very encouraging, current CAR T cell therapies have not shown the same level of success in targeting solid tumors and alternative approaches are required to achieve clinical efficacy in solid tumor patients. We describe a combinatorial approach whereby targeted gene deletion and transgene insertion occur simultaneously resulting in more potent CAR T cells for solid tumor applications. Immunomodulatory cytokines can stimulate vigorous antitumor responses and are candidates for increasing CAR T cell efficacy in solid tumors. However, the clinical application of cytokine therapy has been limited by systemic toxicity, particularly for strong effector cytokines such as IL-12. Limiting IL-12 expression to within the tumor microenvironment may reduce unwanted toxicity while enhancing CAR T cell functionality. Immune checkpoint gene programed cell death 1 (PDCD1) is a regulator of T cell functionality that is highly upregulated following T cell activation, with antibody and nuclease-mediated inactivation of the PD-1 signaling pathway having been shown to enhance CAR T cell functionality. Here, we used megaTAL genome editing/homology directed repair (HDR) to place an IL-12 transgene under the control of the PDCD1 promoter, linking IL-12 production with CAR T cell activation as well as eliminating PD-1 expression. CAR expression was combined with site specific transgene expression as follows: lentiviral vector-engineered T cells were treated with a PDCD1-specific megaTAL and transduced with adeno associated virus-6 (AAV6) containing a promoter-less IL-12 transgene flanked by PDCD1 homology regions. We observed highly efficient HDR, with inducible IL-12 expression from the endogenous PDCD1 promoter being dependent on T cell activation. Minimal IL-12 production was detected under resting conditions, whereas PMA/Ionomycin or co-culture with CAR+ target cell lines resulted in higher IL-12 secretion. Expression of IL-12 under the PDCD1 promoter enhanced CAR T cell cytokine production and cytotoxicity, especially under conditions of repeated antigen exposure. In summary, we describe a novel genome editing strategy to enhance CAR T cell functionality. Using HDR, we were able to engineer CAR T cells to simultaneously disrupt the PDCD1 gene and place a potentially therapeutic transgene under inducible transcriptional control. The IL-12/CAR T cells exhibited activation-dependent IL-12 production and enhanced cytokine and cytotoxicity responses against tumor cells in vitro. HDR may represent a promising approach to enhance CAR T cell functionality in solid tumor applications. Citation Format: Baeckseung Lee, Wai-Hang Leung, Jasdeep Mann, Kyle Havens, Joel Gay, Richard A. Morgan, Alexander Astrakhan. Enhancing CAR T cell activity by linking IL-12 expression to the endogenous PDCD1 promoter [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 1536.