Abstract 28: A CRISPR/Cas9 screening platform ImmuT Finder® enables in vivo T cell functional screening and identification of potent immunoregulatory targets for next-generation T cell therapy

Abstract Insufficient tumor infiltration and the presence of various immunosuppressive mechanisms in tumor microenvironment (TME) are two major challenges compromising the anti-tumor efficacy of T cell therapy post infusion. PD-1/PD-L1 blockade has been proven to benefit a portion of late-stage solid tumor patients, however KO of PD-1 may impact the long-term persistence of CAR-T cells, indicating target selection strategies for gene editing of cell therapy products may differ from antibody drugs. To discover potent and/or novel immunoregulatory targets that could maximize the function of T cell therapy, we established an in vivo CRISPR/Cas9 screening platform, ImmuT Finder®, to identify functional gene targets in primary mouse T cells. Notably, T cells carrying sgRNA library have been collected at different harvest time points post transfer based on different screening purposes; library size has been rationally designed to ensure good coverage of the whole library in harvest samples. We selected top targets from in vivo screening and quickly validated their function in primary human T cells in a 96-well array. Then, the top targets that showed significant enhancement in proliferation and function of human T cells were further selected, and their combinational effects on human peripheral T cells and TIL were tested both in vitro and in vivo. Finally, GT307 stands out as the most potent KO combination, which significantly improves serial tumor killing of TCR-T and CAR-T as well as autologous tumor cytotoxicity of TIL in vitro. Furthermore, GT307 KO TCR-T and TIL demonstrated significantly improved tumor control in mouse models than their unedited counterparts and promoted long-term persistence with low support of IL-2. In summary, these data demonstrated that our ImmuT Finder® platform enables discovery of potent and/or novel targets for the development of T cell product and supports the clinical assessment of GT307 as a next-generation T cell therapy. Citation Format: Jingwei Sun, Jiahui Jin, Jingman Wang, Yao Sheng, Fei Li, Lingyun Chen, Yongchao Tan, Zhao Xu, Yanbin Liu, Ke Liu, Yarong Liu. A CRISPR/Cas9 screening platform ImmuT Finder® enables in vivo T cell functional screening and identification of potent immunoregulatory targets for next-generation T cell therapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 28.