Vaccine-primed CAR T-cells reject antigen-heterogenous tumors via host immunity

Chimeric Antigen Receptor (CAR) T-cell therapy effectively treats human cancer, but loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T-cells triggers engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR-T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited priming of endogenous anti-tumor T-cells (antigen spreading). This process was accompanied by shifts in CAR-T metabolism toward oxidative phosphorylation and was critically dependent on CAR T-derived IFN-γ. Antigen spreading induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR-antigen-negative, and heterogenous tumor control was further enhanced by genetically amplifying CAR T IFN-γ expression. Thus, CAR T-cell-derived IFN-γ plays a critical role in promoting antigen spreading, and vaccine boosting provides a clinically-translatable strategy to drive such responses against solid tumors. Endogenous CD45.2+ CD4 and CD8 T cells were sorted with fluorescence activated cell sorting (FACS) collectively into 1x RPMI +10%FBS/