RNA-seq analysis of human CD19 CAR T cells overexpressing RAB5

Chimeric antigen receptor (CAR) T cells (CARTs) are engineered T cells designed to target specific molecules on the surface of cancer cells. Using a continuous antigen exposure (CAE) assay to model this interaction, we observed that tumors can capture CAR molecules, leaving the CARTs devoid of surface-expressed CARs and unable to kill tumors after repeated challenges. Overexpression of Rab5, a key regulator of endocytosis, prevents the loss of CARs and enhances CARTs activity. Rab5 significantly increased the endocytic recycling of T cells without causing other perturbations to CART function. Interestingly, we observed membrane protrusions on the CART cell surface, which disappeared after multiple tumor challenges. Rab5 overexpression maintained these protrusions after multiple tumor engagements, and the presence of membrane protrusions correlated with tumor clearance. In vivo, Rab5-expressing CARTs demonstrated improved activity and were able to clear an otherwise recalcitrant mesothelin-expressing solid cancer. These findings suggest that engineering CARTs with Rab5 overexpression could enhance the clinical efficacy of CAR T cell therapy.