CD33 KO -CD33-mesothelin loop CAR design avoids fratricide and improves efficacy of iNK cells against acute myeloid leukemia
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Acute myeloid leukemia (AML) patients are often older, which brings challenges of endurance and persistent efficacy of autologous CAR-T cell therapies. Allogenic CAR-NK cell therapies may offer reduced toxicities and enhanced anti-leukemic potential against AML. In this study, we designed a novel CD33-mesothelin loop CAR (Loop CAR) and evaluated its anti-tumor efficacy in human umbilical cord blood-derived NK (UCB-NK) cells and human pluripotent stem cell-derived NK (hPSC-iNK) cells. The Loop CAR exhibited superior cytotoxicity against dual-antigen-positive tumor cell lines and primary AML cells. To further avoid fratricide caused by endogenous CD33 expression in NK cells, we established a hPSC-derived cell line via knockout of CD33 gene (CD33 KO ) and engineered Loop CAR. Rather than enforced expression of exogenous CD16, we generated abundant mature CD33 KO -Loop CAR-iNK cells highly expressing endogenous CD16 via an organoid induction approach. This innovative strategy effectively mitigated NK cell fratricide and significantly enhanced CD33 and mesothelin-mediated specific cytotoxicity. Moreover, the CD33 KO -Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mice and significantly prolonged survival. hPSC-derived CD33 KO -Loop CAR-iNK cells possess unique advantages and translational potential for treating AML.