Dual-modular-nanobody CAR-T cell technical platform against the solid tumor microenvironment

BACKGROUND: CAR-T therapy is effective in hematologic cancers but faces challenges in solid tumors due to antigen heterogeneity and an immunosuppressive tumor microenvironment (TME). Systemic CTLA-4 blockade enhances immunity but often causes severe adverse events. To overcome these limitations, we developed a dual-modular nanobody-based CAR-T platform targeting fibroblast activation protein (FAP) on cancer-associated fibroblasts and locally releasing an anti-CTLA-4 nanobody within the tumor stroma. METHODS: FAP/CTLA-4 dual-module CAR-T cells were generated and assessed in vitro for antigen-specific cytotoxicity, cytokine release, and exhaustion. Antitumor efficacy was evaluated in xenograft models, measuring tumor growth, survival, and T-cell infiltration (Ethics Approval Number: 202001011). One patient with refractory glioblastoma received intrathecal infusion; clinical response, cerebrospinal fluid (CSF) cytokines (Ethics Approval Number 2022-0553-01), and safety were monitored. Tumor and immune microenvironment changes were analyzed via transcriptomic sequencing and multiplex immunofluorescence staining. RESULTS: In vitro, engineered CAR-T cells showed potent cytotoxicity, cytokine production, and reduced exhaustion. In vivo, they induced tumor regression, prolonged survival, and increased T-cell infiltration. In the glioblastoma patient, intrathecal administration resulted in disease stabilization, elevated CSF cytokines, and a favorable safety profile. Transcriptomic sequencing and multiplex immunofluorescence staining indicated TME remodeling toward an immunologically active state. CONCLUSIONS: FAP/CTLA-4 DMN CAR-T overcomes the immunosuppressive solid tumor microenvironment through localized immunomodulation, demonstrating promising efficacy in preclinical models and a patient with refractory glioblastoma.