Abstract 4804: RNA binding proteins in CAR-T armoring: Reprogramming T cells for tumor killing

Background: CAR T cell therapy uses genetically engineered T cells to express a chimeric antigen receptors (CARs) that trigger detection and killing of target tumor cells. It has been previously shown that a functional screen of mouse RNA binding proteins (RBPs) identifies genes that regulate the abundance of activated plasma cells. Deletion of specific RBP genes was shown to increase the potency of effector CD8 T cell functions. We hypothesized that these observations can be translated as an armoring approach to improve human CAR-T cell functions. Methods: We performed cytotoxicity assays based screening using Her2 CAR T cells with or without 46 RBP knockouts in co-culture with Her2 expressing target cells. The co-culture were set up at a low E:T ratio of 1:32 to evaluate the effect of RBP KO on CAR-T cell exhaustion through engagement in multiple rounds of target cell killing. We validated the modulation of killing with time-course experiments using several Her2 cell lines as HCC-1954, SKOV-3 as well as a control Her2-KO HCC-1954 cell line. We further evaluated the relevance of these findings using patient derived organoid models co-cultured with CAR-T cells bearing specific RBP KOs. Results: Our screen identified several RBPs which upon deletion enhanced Her2 CAR T killing similarly to Med12 deletion which was previously identified as a CAR-T cell armouring candidate. NGS sequencing and FACS analysis validated the KO efficiencies in CAR Ts used for co-culture. Intracellular flow cytometry prior to co-culturing showed that certain CAR T KOs exhibited higher expression of Interferon gamma, Granzyme B and Perforin 1. RBP KOs in CAR Ts also showed increased killing of patient derived organoids in co-culture. Conclusion: Our findings suggest that KO of RBPs may be a good strategy to enhance CAR-T cell cytotoxic function and persistence. They suggest the importance of RBPs as a novel class of armoring targets for CAR-T cell enhancement. Citation Format: Augustin B. Biswas, Martin Turner, Romain Lara. RNA binding proteins in CAR-T armoring: Reprogramming T cells for tumor killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4804.