Concurrent targeting of tumors and their microenvironment using CAR-T cells specific for fibroblast activation protein alpha

Chimeric antigen receptor T (CAR-T) cell therapy for solid tumors is challenged by the immunosuppressive tumor microenvironment (TME) and physical barriers formed by Fibroblast Activation Protein alpha (FAPalpha)-expressing cancer-associated fibroblasts (CAFs). Furthermore, high-affinity CAR-T cells often induce trogocytosis and T cell exhaustion, leading to target antigen loss and limiting persistence. We sought to develop FAPalpha CAR-T cells with optimized binding affinity to overcome these obstacles. We generated FAPalpha CAR-T cells (CD28/CD3ζ) by shuffling new light chains fused to the heavy chain of the parental clone of FAPalpha CAR using a high-throughput scFv library T-cell system (Eumbody System). We identified 3 new clones using this method, FL1, FL8 and FL12, exhibiting slightly lower structural avidity compared to the parental clone. We investigated their function in vitro and in vivo using glioblastoma cell lines. Unlike the parental clone FAPalpha-CAR-T, the optimized FL1, FL8 and FL12 clones preserved FAPalpha expression on target cells, demonstrating successful mitigation of trogocytosis, which was inversely correlated with structural avidity. In in vitro cytotoxicity assays, FL12 CAR-T cells showed superior real-time killing against FAPa-positive U87 cells and better suppression of U87 spheroid growth, while exhibiting a less exhausted T-cell phenotype. In vivo, FL12 CAR-T cells effectively suppressed U87 tumor growth in a subcutaneous xenograft model, with statistically significant improvements compared to both conventional scFv-CAR-T cells and negative control T cells even at low cell doses. To further evaluate their efficacy, FL12 CAR-T cells are currently being tested in in vitro cytotoxicity assays using 3D mixed tumor spheroid (U87 or U251 plus CAFs). This work is designed to confirm their ability to be highly cytotoxic against FAPalpha-positive CAFs, successfully migrate into mixed tumor spheroids, and achieve a significant reduction in tumor size when compared with conventional scFv-CAR-T cells and tumor-only spheroids. In conclusion, scFv-optimization successfully generated FAPalpha CAR-T cells that mitigate trogocytosis and exhibit superior phenotype, persistence, and anti-tumor efficacy. This strategy allows for concurrent targeting of solid tumor cells and the CAF-rich microenvironment, representing a promising and readily translatable approach for refractory solid malignancies.