UCSF

Tumors drive T cell exhaustion in both tumor-infiltrating lymphocytes and CAR T cells, marked by increased inhibitory receptor expression, impaired cytotoxicity, and reduced cytokine production, ultimately limiting durable clinical responses. In this work, I have performed a high-density base editor screen of A20/TNFAIP3, a multifunctional ubiquitin-modifying protein, to map variants to T cell functions. In addition, we performed genome-wide loss-of-function screening in repetitively stimulated human T cells and identified the multifunctional ubiquitin-modifying protein A20/TNFAIP3 as a major negative regulator of exhausted T cell persistence. Protein large language modeling, deep base-editing mutagenesis, and studies in immunocompetent mice with domain-specific inactivating mutations revealed A20’s non-enzymatic M1 ubiquitin-binding zinc finger 7 (A20ZF7) motif as critical to suppression of anti-tumor immunity. A20ZF7-deficient CD8+ tumor-infiltrating lymphocytes (TILs) resisted terminal exhaustion and circumvented an unappreciated mechanism restraining perforin degranulation in terminally exhausted cells. Human chimeric antigen receptor (CAR)-T cells engineered via base-editing to inactivate A20ZF7 via a single missense mutation also resisted exhaustion, secreted more perforin and robustly suppressed cancer in vivo. These studies pinpoint A20ZF7 as a novel T cell checkpoint and reveal precision base-editing of missense mutations as an effective approach to enhance CAR-T cell therapy.