Next-generation CAR-T cells that target only tumor stroma and matrix: Collagen-binding IL-12-expressing FAP-CAR-T show strong anti-tumor efficacy but are safe

Fibrotic tumor subtypes have been identified across multiple solid malignancies, including pancreatic, breast, lung, and colorectal cancers. These fibrotic tumors are generally difficult-to-treat and are characterized by extensive desmoplastic stroma and pronounced heterogeneity. The fibrotic tumor microenvironment (TME) not only serves as a physical barrier that impedes therapeutic penetration and immune cell infiltration but also contributes to therapeutic resistance, rendering these cancers particularly challenging to treat. Cancer-associated fibroblasts (CAFs), which are abundant in the TME, have emerged as a promising therapeutic target. In this study, we developed a novel chimeric antigen receptor T cell (CAR-T) therapy specifically targeting fibroblast activation protein (FAP) expressed on CAFs. After binding to FAP, NFAT signalling enables the subsequent local secretion of collagen-binding domain-fused interleukin-12 (CBD-IL-12) from CAR-T cells. This CAR-T cells only remove CAF but also concentrate collagen-binding IL-12 in the tumor to enhance host immune activation within the tumor microenvironment. The CBD-IL-12 expressing FAP CAR-T cells exhibited effective cytotoxicity against fibroblast cell lines in vitro. In vivo, CBD-IL-12 expressing FAP CAR-T cells significantly suppressed tumor progression of murine breast cancer and pancreatic cancers, which were better than 2nd generation FAP CAR-T or wt IL-12-expressing CAR-T. Importantly, CBD-fusion to IL-12 in the CAR-T design improved accumulation of IL-12 within tumor tissues >10 times, whereas minimizing systemic IL-12 exposure and reducing associated toxicities. Immunophenotyping revealed increased infiltration of CD8⁺ cytotoxic T lymphocytes and reduced regulatory T cells (Tregs) in the TME of mice treated with CBD-IL-12-expressing FAP CAR-T, compared to those treated with FAP CAR-T alone or with wt IL-12 secretion. This dual-function CAR-T approach serves to first deplete CAFs, thereby disrupting the physical barrier impeding immune cell infiltration, and second, to activate anti-tumor immunity through localized IL-12 delivery. We have created a new therapeutic concept, which utilizes CAR-T cells for non-tumor antigen target, but still activates immune cells locally in the tumors, which could be a universal purpose CAR-T cells for multiple cancer types, especially for fibrotic or post-x-ray tumors.