Abstract A51: Rationally improving T cell-mediated cancer immunotherapies using Sleeping Beauty mutagenesis to identify novel therapeutic targets
Authors: Rogers Laura M. , Dupuy Adam J. , Weiner George J. 
Published: June 2021

Expanding immunotherapy success to a broader patient population is a top priority in the field, and T-cell infiltration into tumors appears to be an important prerequisite for the success of both immune checkpoint blockade and chimeric antigen receptor T cell (CAR-T) therapies. The data presented here are results from multiple genetic screens designed to identify T-cell genes that promote their intratumoral infiltration. The gene candidates we discovered may be leveraged to enhance T cell-mediated cancer immunotherapies, with particular translational potential for CAR-T therapies. We designed a unique forward genetic screen approach to identify T-cell genes that contribute to intratumoral T-cell accumulation using Sleeping Beauty mutagenesis in T cells. Sleeping Beauty has a unique advantage over CRISPR and shRNA screens because it allows us to induce gain-of-function mutations, in addition to loss-of-function mutations, across the entire genome. Our systematic screen approach was performed in immunocompetent murine models of melanoma and lymphoma that preserve the complexity of the tumor microenvironment. Further, we carried out additional screens where mutagenized mice were treated with immune checkpoint inhibitor anti-PD-1 in order to assess how the selective pressures of this therapy influence the mutation spectra. Our screens identified 368 genes expected to impact intratumoral T-cell infiltration. Some of the candidate genes we identified were previously found to directly regulate intratumoral CD8+ T cell infiltration (e.g., IL-23R), and the candidate gene list was significantly enriched for T cell anergy genes (P = 0.0074). Fifty-one genes were mutated in more than one mouse and represent strong gene candidates for functional validation. Importantly, none of the classical immune checkpoint molecules were identified in our screens, demonstrating the novelty of our results since these genes likely are not currently being investigated as drug targets to enhance immunotherapy. We hypothesize that the biologic functions of these genes have the potential to modify important T-cell functions including trafficking to the tumor, clonal expansion, and sustained viability once inside the tumor. Interestingly, anti-PD-1 treatment had a profound effect on candidate gene selection. One gene in particular was much more frequently mutated in T cells from mice treated with anti-PD-1 therapy than untreated mice. In a series of validation experiments, we demonstrate that modulating one of these gene candidates with a small-molecule inhibitor impacts tumor growth and CD8+ T-cell infiltration in mice when combined with anti-PD-1 therapy. From this, we conclude that this screen approach is a viable way to discover novel immunotherapy targets. Current studies are exploring potential mechanisms by which this candidate gene impacts T-cell function and assessing therapeutic impact of overexpression in a CAR-T system.

Citation Format: Laura M. Rogers, Adam J. Dupuy, George J. Weiner. Rationally improving T cell-mediated cancer immunotherapies using Sleeping Beauty mutagenesis to identify novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A51.