Discovery and functional optimization of a fully human VH domain-based anti-FAP CAR-T cell therapy with enhanced persistence and safety

Chimeric antigen receptor T-cell (CAR-T) therapiey targeting disease-associated stromal cells represent an emerging strategy for treating pathological conditions characterized by fibroblast activation. Fibroblast activation protein (FAP), a cell-surface serine protease selectively expressed on activated fibroblasts, including cancer-associated fibroblasts (CAFs) and fibroblast populations involved in fibrotic diseases, has become an attractive therapeutic target. However, the therapeutic performance of FAP-targeted CAR-T cells remains highly dependent on the optimization of antigen-recognition domains, and it remains unclear which anti-FAP binder optimally supports CAR-T effector function. This study aimed to address this gap by establishing a functional screening platform to identify optimized anti-FAP VH domain sequences and to systematically compare them with the clinically evaluated F19 CAR as a benchmark. In this study, we established a functional screening platform to identify optimized anti-FAP heavy-chain variable (VH) domain sequences for CAR-T engineering. Using phage display technology, novel anti-FAP VH binders were isolated and compared with the clinically evaluated F19 antibody-derived CAR as a benchmark. Candidate CAR-T cells were systematically evaluated using antigen-specific activation assays, cytotoxicity analysis, real-time killing kinetics, serial antigen stimulation assays, T-cell phenotype characterization, cytokine profiling, and in vivo efficacy and safety studies using FAP-expressing fibroblast and tumor models. Among the identified candidates, A04 VH-based CAR-T cells exhibited superior functional characteristics compared with F19 CAR-T cells, including enhanced antigen-specific cytotoxicity, accelerated target-cell elimination, and sustained effector function after repeated antigen stimulation. A04 CAR-T cells maintained a higher proportion of stem cell-like memory T cells, reduced exhaustion-associated marker expression, and a favorable CD8/CD4 T-cell composition. In FAP-expressing fibroblast models, A04 CAR-T cells demonstrated selective and potent elimination of fibroblasts without detectable off-target activity. Furthermore, in FAP-positive tumor models, A04 CAR-T cells achieved significant tumor suppression, prolonged peripheral persistence, controlled cytokine release, and minimal systemic toxicity. This study establishes a functional screening-driven strategy for developing optimized FAP-targeted CAR-T cells and identifies A04 VH-based CAR-T cells as a promising therapeutic candidate for diseases driven by pathological fibroblasts expressing FAP. Beyond tumor-associated stromal targeting, this platform offers a potential framework for CAR-T therapy in broader fibroblast-mediated disorders, including fibrotic diseases.