FMNL1 overexpression enhances tumor-specific TIL and CAR-T cell accumulation and therapeutic efficacy in solid tumors

Purpose: Adoptive T cell therapies such as tumor-infiltrating lymphocytes (TIL) and chimeric antigen receptor (CAR)-T cells show limited efficacy against solid tumors. This is due in part to physical barriers in solid tumors, including abnormal vasculature and dense extracellular matrix, which limit T cell infiltration and persistence within restrictive tumor microenvironments. We investigated whether overexpression of Formin-like-1 (FMNL1), a cytoskeletal regulator of T cell migration, could overcome these physical barriers and improve T cell accumulation and therapeutic activity in solid tumors. Methods: We engineered tumor-specific TILs and CAR-T cells to overexpress FMNL1 using a bioengineering platform. We first assessed control and FMNL1-overexpressing T cell activation, cytotoxicity, and restimulation in vitro. Then using murine tumor models, we evaluated tumor infiltration and therapeutic efficacy of adoptively transferred control and FMNL1-overexpressing TILs and CAR-T cells in melanoma and lung carcinoma models. Results: FMNL1-overexpressing T cells maintained normal activation and tumor cell killing compared to control T cells. In vivo, FMNL1 overexpression significantly increased TIL and CAR-T cell accumulation at melanoma and lung carcinoma solid tumor sites compared to controls. Importantly, adoptive transfer of FMNL1-overexpressing CAR-T cells prolonged survival in melanoma tumor-bearing mice relative to control CAR-T cells, in both immunodeficient and immunocompetent recipient mice. Conclusions: This is the first demonstration that enhancing cytoskeletal dynamics via FMNL1 overexpression increases T cell accumulation in solid tumors and improves therapeutic efficacy. These findings highlight a novel, tumor antigen and CAR construct independent strategy to overcome physical barriers in the tumor microenvironment and suggest translational potential for improving CAR-T cell therapy in patients with solid tumors.