Rab5 Overcomes CAR T Cell Dysfunction Induced by Tumor-Mediated CAR Capture

Continuous interaction between chimeric antigen receptor (CAR) T cell (CART) and tumors often result in CART dysfunction and tumor escape. We observed that tumors can take up CAR molecules, leaving CARTs without surface-expressed CARs and thus unable to kill tumors after prolonged exposure. Overexpression of Rab5 resulted in augmented clathrin-independent endocytosis, preventing loss of surface-expressed CARs, and enhanced CART activity. Interestingly, we observed membrane protrusions on the CART cell surface which disappeared after multiple tumor challenges. Rab5 maintained these protrusions after repeated tumor engagements and their presence correlated with effective tumor clearance, suggesting a link between endocytosis, membrane protrusions, and cytolytic activity. In vivo, Rab5-expressing CARTs demonstrated improved activity and were able to clear an otherwise refractory mesothelin-expressing solid cancer in humanized mice by maintaining CAR surface expression within the tumor. Thus, pairing Rab5 with CAR expression could improve the clinical efficacy of CART therapy. Highlights O_LI"CAR-jacking" occurs when surface CAR is internalized by target tumor cells. C_LIO_LIRab5 overexpression prevents "CAR-jacking" and enhances CART function. C_LIO_LIRab5 promotes CAR endocytic recycling and maintains membrane protrusions. C_LIO_LIRab5-expressing CARTs exhibit enhanced therapeutic efficacy against solid tumors. C_LI