Base-editing a single missense mutation in A20 enhances CAR-T cell efficacy

T cell exhaustion limits the efficacy of cancer immunotherapies. Here, we performed genome-wide loss-of-function screening in repetitively stimulated human T cells and identified the mulitfunctional 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 (A20 ^ZF7 ) motif as critical to suppression of anti-tumor immunity. A20 ^ZF7 -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 A20 ^ZF7 via a single missense mutation also resisted exhaustion, secreted more perforin and robustly suppressed cancer in vivo . These studies pinpoint A20 ^ZF7 as a novel T cell checkpoint and reveal precision base-editing of missense mutations as an effective approach to enhance CAR-T cell therapy.