CD33 ^KO -CD33-mesothelin loop CAR design avoids fratricide and improves efficacy of iNK cells against acute myeloid leukemia

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.