Autocrine PD-1-blocking nanobodies enhance the antitumor efficacy of TCR-like CAR-T cells by attenuating t cell exhaustion.

BACKGROUND: Chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in hematologic malignancies, but its efficacy against solid tumors is severely limited by two core barriers: the scarcity of uniformly expressed tumor-associated antigens (TAAs) and the immunosuppressive tumor microenvironment (TME), in which PD-1/PD-L1 signaling promotes irreversible T cell exhaustion. This study aimed to develop a novel T cell receptor-mimic (TCRm) CAR-T platform with autocrine anti-PD-1 nanobody secretion to simultaneously overcome antigen heterogeneity and TME immunosuppression. METHODS: A lentiviral vector encoding a TCRm CAR targeting HLA-A*02:01-restricted WT1 and a secreted anti-PD-1 nanobody was constructed to generate WT1-CAR-Nb T cells. WT1-CAR T cells (CAR-only control) and Mock T cells were also prepared. In vitro assays included antigen-specific cytotoxicity, proinflammatory cytokine (IFN-γ, TNF-α) detection, chronic antigen stimulation-induced T cell exhaustion model, and transcriptomic analysis. In vivo efficacy was evaluated in human ovarian cancer SKOV3-A2 xenograft mice via tumor growth monitoring, survival analysis, intratumoral T cell infiltration assessment, and safety evaluation. RESULTS: WT1-CAR-Nb T cells targeting the HLA-A*02:01-restricted WT1 and constitutively secreting anti-PD-1 nanobody were successfully generated. In vitro functional assays demonstrated that WT1-CAR-Nb T cells exhibited significantly enhanced antigen-specific cytotoxicity and secreted markedly higher levels of proinflammatory cytokines IFN-γ and TNF-α compared with conventional WT1-CAR T cells upon specific antigen stimulation. Critically, autocrine PD-1 nanobody blockade effectively alleviated TME immunosuppression, as reflected by downregulated expression of multiple exhaustion markers (TIM-3, CTLA-4, TIGIT) and preserved proliferative potential under chronic antigen stimulation. In the SKOV3-A2 solid tumor xenograft model, WT1-CAR-Nb T cells mediated superior tumor growth control and significantly prolonged overall survival, which was closely correlated with enhanced intratumoral T cell infiltration and persistent in vivo expansion of engineered T cells. CONCLUSION: Local autocrine PD-1 checkpoint blockade potently enhances the effector function and durability of TCRm CAR-T cells. This armored TCRm CAR-T strategy is a promising therapeutic approach for solid tumor immunotherapy, with broad translational potential for other TCRm CAR systems targeting distinct peptide-MHC epitopes.