Synthetic reprogramming of gammadelta CAR T cells with the LTBR gene optimizes function in the TME and overcomes effector limitations

Cell therapies have transformed outcomes in hematologic cancers, yet their efficacy in solid tumors remains limited by the profoundly immunosuppressive tumor microenvironment (TME), which employs multiple, overlapping mechanisms to suppress immune activity. Current armoring strategies address these barriers individually, leaving key vulnerabilities, while autologous approaches remain costly and difficult to scale. Allogeneic gammadelta CAR T cells represent an attractive, off-the-shelf platform, but their persistence and potency within the solid tumor TME require enhancement. To advance scalable and effective solid tumor therapies, we pursued a data-driven strategy to optimize gammadelta T cell biology while increasing TME resilience. Owing to limited knowledge of gammadelta T cell functional states in antitumor response, we implemented an empirical approach integrating genome-scale discovery and AI-driven analytics to uncover pathways governing gammadelta CAR T function. We conducted a genome-wide overexpression screen to identify reprogramming genes that protect CAR T cells from adenosine, TGF-beta, immunosuppressive macrophages, and regulatory T cells while improving persistence. Among 12,000 genes screened, 51 candidates conferred protection against three or more suppressive mechanisms. Lymphotoxin beta receptor (LTbetaR)-a TNF superfamily receptor not normally expressed in T cells-emerged as the top hit, reprogramming T cells into highly proliferative, persistent, and multifunctional states, as confirmed by bulk and single-cell RNA-seq. In high-stress assays, LTbetaR expression conferred broad resistance to immunosuppression, including complete abrogation of Treg-mediated inhibition. In gammadelta CAR T cells, LTbetaR induced an alphabeta-like effector program characterized by enhanced cytotoxicity, persistence, and cytokine modulation. scRNA-seq revealed new mechanisms of gammadelta CAR T exhaustion and effector differentiation distinct from alphabeta CAR T cells. In vivo, LTbetaR-armored alphabeta and gammadelta CAR T cells targeting CLDN6 mediated complete, durable regressions in an aggressive ovarian cancer model where controls were ineffective. Finally, we established a GMP-compatible manufacturing process tailored to gammadelta T cell biology, producing highly pure, potent cells suitable for multi-patient allogeneic use. Together, this work defines a comprehensive next-generation engineering and manufacturing framework that overcomes gammadelta CAR T cell limitations and enables durable activity in solid tumors.