Multiplexed Shrnas Targeting FAS and PTPN2 Enhance CAR T Persistence and Anti-Tumor Efficacy

Cell therapies have shown limited efficacy in solid tumors, in part due to limitations in CAR T cell survival, expansion, and resistance to immunosuppressive pathways operating in the tumor microenvironment (TME). To overcome these hurdles, we screened a library of 171 pair-wise gene knockouts using CRISPR-Cas9 to identify gene pairs that confer greater proliferation and persistence with repetitive stimulation. Combined KO of FAS and PTPN2 led to a significant improvement in T cell effector function and proliferative capacity. FAS, the receptor for FASLG, is highly expressed on patient-derived T cells and has been shown to induce T cell apoptosis and limit T cell persistence in the TME. PTPN2 is a phosphatase, in which deletion increases T cell proliferation and cytotoxicity. To develop this clinically, we generated CAR T cells with a shRNA module capable of multiplexed knockdown of both FAS and PTPN2. The FAS/PTPN2 shRNA module protected T cells from FAS-mediated apoptosis and resulted in a marked increase in T cell expansion over the course of a repetitive stimulation assay. Consistent with these observations, RNAseq analysis revealed enhanced transcriptome-wide signatures of cell cycle and T cell effector function. Finally, in a tumor xenograft model, CAR T cells containing FAS/PTPN2 shRNA module demonstrated significantly improved tumor control in addition to increased cell numbers in circulation compared with CAR T cells expressing an irrelevant shRNA module. The use of a multiplexed shRNA module is a powerful approach to improve T cell intrinsic functionality in cellular therapies, enabling: 1) tailored levels of inhibition of multiple key regulators of T cell biology with constitutive or antigen-gated expression, and 2) a method for genetic perturbations in T cells with reduced risk of toxicity or transformation compared with multiple DNA edits. The FAS/PTPN2 shRNA module described here will be tested as a component of AB-1015, an integrated circuit T cell therapy for solid tumors expressing ALPG and mesothelin. Citation Format: John Gagnon, Adam J. Litterman, Jason A. Hall, Dina Polyak, Stanley Zhou, Sahil Joshi, Oliver Takacsi-Nagy, Hans Pope, luisa silva, brenal K. Singh, jeffrey M. Granja, david DeTomaso, Edgar Aristil-Lepe, Michelle Tan, Brendan Galvin, Grace X. Zheng, Stephen Santoro, Aaron Cooper, Natalie Bezman, W Nicholas Haining. Multiplexed shRNAs targeting FAS and PTPN2 enhance CAR T persistence and anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2830.