Membrane-proximal binding of PSMA facilitates synapse formation with CAR and enhances antitumor activity of PSMA CAR-T cells against prostate cancer

BACKGROUND: Prostate-specific membrane antigen (PSMA) represents an ideal therapeutic target for prostate cancer. However, the clinical efficacy of PSMA-targeted chimeric antigen receptor T (CAR-T) cell therapy remains unsatisfactory. Strategies to enhance anti-tumor activity-such as optimizing CAR antigen-binding design-are critical for maximizing the clinical benefits of this treatment. METHODS: We constructed CARs targeting membrane-proximal (MP) and membrane-distal (MD) epitopes of PSMA to evaluate how epitope location influences CAR-T cell anti-tumor function. We systematically evaluated the antitumor efficacy of MP CAR-T and MD CAR-T cells both in vitro and in xenograft mouse models in vivo. Furthermore, by integrating transcriptomic and proteomic datasets, we postulated the underlying antitumor mechanism of MP CAR-T cells. RESULTS: Despite comparable antigen affinity and phenotypic profiles, MP CAR-T cells demonstrated superior in vitro cytotoxicity and inflammatory cytokine secretion. In vivo, MP CAR-T cells exhibited significantly stronger control of tumor growth and prolonged survival in both solid tumor and bone metastasis models. Transcriptomic and proteomic analysis revealed that MP CAR-T cells had enhanced activation of immune synapse-related pathways and a unique upregulation of the STING-mediated interferon signaling pathway. CONCLUSION: Targeting the proximal membrane protease domain of PSMA generates functionally superior CAR-T cells. The enhanced efficacy is associated with more robust immunological synapse formation and the concomitant activation of the STING innate immune pathway, offering a novel mechanistic insight for optimizing CAR-T cell design.