Postdoc Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences Guangzhou, Guangdong, China (People's Republic)
Abstract: CAR-NK cell therapy has been investigated for treating acute myeloid leukemia (AML). CD33 is a well-established therapeutic target for AML. Conventional CD33 CAR-NK cells are prone to fratricide due to endogenous expression of CD33 on expanded NK cells. Mesothelin (MSLN), a tumor differentiation antigen, is expressed in approximately 36% of pediatric AML cases and 14% of adult AML cases. In this study, we designed a novel CD33-MSLN 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 optimize this approach, we established a hPSC-derived cell line via knockout of endogenous CD33 gene (CD33 KO) and integration of the Loop CAR construct. Using organoid induction technology, we generated mature CD33KO-Loop CAR-iNK cells, characterized by expression of endogenous CD16. This innovative strategy effectively mitigated NK cell fratricide and significantly enhanced CD33-mediated specific cytotoxicity. Moreover, the CD33 KO-Loop CAR-iNK cells demonstrated superior tumor-killing activity in AML xenograft mice and significantly prolonged survival of the treated animals. Our findings provide evidence supporting that hPSC-derived CD33 KO-Loop CAR-iNK cells have translational potential for treating AML.
