Abstract C008: <i>NFIL3</i> emerges as a driver of CAR T cell dysfunction from integrated in vivo/in vitro CRISPR screen

Abstract CAR therapy has transformed the treatment landscape for hematological malignancies but success in solid tumors is at present limited. One important mechanism underlying treatment failure is insufficient functional persistence of the engineered T cells. To overcome the functional decline arising from chronic antigen exposure, we conducted a multi-parameter in vivo screen targeting a curated set of 400 transcription factors. These transcription factors were selected based on their involvement in T cell exhaustion across diverse contexts—including murine acute and chronic stimulation models, CAR designs associated with enhanced persistence, and transcriptional signatures from patients achieving durable clinical responses following CAR therapy. We employed the acute lymphoblastic leukemia (B-ALL) NALM6 mouse model and introduced two sequential tumor rechallenges immediately after primary tumor clearance to simulate persistent CAR activation. The in vivo screen was performed as an enrichment-based approach to identify gRNAs that enhance CAR T cell persistence under chronic in vivo stimulation. Top candidates identified in the in vivo screen were subsequently evaluated in an in vitro chronic activation screen where they were evaluated for multiple functions under persistent activation – expansion, cytotoxicity, differentiation, and inhibitory receptor expression. This screen revealed NFIL3 as a key negative regulator of CAR T cell function. Genetic ablation of NFIL3 in CAR T cells promoted sustained expansion, enhanced IL-2 and Th2-associated cytokine production, and restricted terminal differentiation, overall favoring a transcriptional phenotype that has been associated with superior clinical responses in patients treated with CAR T cells. NFIL3 disruption improved antitumor activity across multiple stress-test models, spanning different CAR designs and tumor types in both, hematologic and solid cancers. These findings underscore the power of comprehensive, multiparametric in vivo genetic screening to uncover novel regulators of CAR T cell function and position NFIL3 as a compelling therapeutic target for enhancing CAR T cell efficacy across diverse cancer indications. Citation Format: Nayan Jain, Yuzhe Shi, Celina May, Sneha Mitra, Philip Bucher, Anton Dobrin, Zeguo Zhao, Sophie Hanina, Vinagolu Rajasekhar, Christina Leslie, Francisco Sánchez-Rivera, Judith Feucht, Michel Sadelain. NFIL3 emerges as a driver of CAR T cell dysfunction from integrated in vivo/in vitro CRISPR screen [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C008.