Flagellin engineering enhances CAR-T cell function by reshaping tumor microenvironment in solid tumors.

Background: Adoptive cell therapy using genetically engineered chimeric antigen receptor (CAR)-T cells is a new type of immunotherapy that directs T cells to target cancer specifically. Although CAR-T therapy has achieved significant clinical efficacy in treating hematologic malignancies, its therapeutic benefit in solid tumors is impeded by the immunosuppressive tumor microenvironment (TME). Therefore, we sought to remodel the TME by activating tumor-infiltrating immune cells to enhance the antitumor function of CAR-T cells.

Methods: We engineered CAR-T cells expressing Salmonella flagellin (Fla), a ligand for toll-like receptor 5, to activate immune cells and reshape the TME in solid tumors. Functional validation of the novel Fla-engineered CAR-T cells was performed in co-cultures and mouse tumor models.

Results: Fla could activate tumor-associated macrophages and dendritic cells, reshaping the TME to establish an "immune-hot" milieu. Notably, this "cold" to "hot" evolution not only improved CAR-T cell function for better control of target-positive tumors, but also encouraged the production of endogenous cytotoxic CD8⁺T cells, which targeted more tumor-associated antigens and were thus more effective against tumors with antigenic heterogeneity.

Conclusion: Our study reveals the potential and cellular mechanisms for Fla to rewire antitumor immunity. It also implies that modifying CAR-T cells to express Fla is a viable strategy to improve the efficacy of CAR-T cell treatment against solid tumors.