Preventing trogocytosis by cathepsin B inhibition augments CAR T-cell function

Abstract Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy in cancer treatment. Nevertheless, most patients receiving CAR T cells relapse within 5 years of treatment. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism in which cell surface proteins transfer from tumor cells to CAR T cells. CMT results in the emergence of antigen-negative tumor cells, which can evade future CAR detection, and antigen-positive CAR T cells, which have been suggested to cause CAR T-cell fratricide and exhaustion. Whether CMT indeed causes CAR T-cell dysfunction and the molecular mechanisms conferring CMT remain unknown. Using a selective degrader of trogocytosed antigen in CAR T cells, we show that the presence of trogocytosed antigen on the CAR T-cell surface directly causes CAR T-cell fratricide and exhaustion. By performing small molecule screening using a custom high-throughput CMT screening assay, we found that the cysteine protease cathepsin B is essential for CMT and that inhibition of cathepsin B is sufficient to prevent CAR T-cell fratricide and exhaustion, leading to improved long-term in vitro and in vivo CAR T-cell persistence and in vitro antitumor activity. Our data demonstrate that it is feasible to separate CMT from cytotoxic activity, that CAR T-cell persistence, a key factor associated with clinical CAR T-cell efficacy, is directly linked to cathepsin B activity in CAR T cells, and that it is possible to improve CAR T-cell function through selective inhibition of CMT.