Enhanced CD33 CAR-NK cells secreting anti-CD73scFv overcome adenosine-mediated immunosuppression and improve anti-AML efficacy

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with dismal outcomes, especially in relapsed/refractory settings. Chimeric antigen receptor natural killer (CAR-NK) cell therapy holds promise but is constrained by the immunosuppressive tumor microenvironment (TME), where adenosine-mediated suppression is a key barrier. OBJECTIVE: To develop a novel CAR-NK construct cotargeting AML cells and the adenosine-rich TME to enhance antileukemia efficacy. METHODS: Ex vivo expanded primary NK cells were used to compare the effects of CD39 versus CD73 blockade on NK cell function via messenger RNA-electroporated antibodies. A CD33-CD73 dual-function CAR-NK construct (integrating CD33-specific lysis and anti-CD73scFv secretion for TME disruption) was designed and transduced into NK cells via retrovirus. Engineered NK cells were characterized for transduction efficiency, expansion, purity, viability, and CAR stability. In vitro cytotoxicity against AML cell lines and primary blasts was assessed, and in vivo efficacy was evaluated in a MOLM-13 xenograft mouse model. RESULTS: CD73 blockade more potently enhanced NK cell activity than CD39 blockade. Retroviral transduction achieved >50% efficiency, and expansion with K562-4-1BBL-mbIL-21/-15 feeder cells yielded NK cells with ≥6,000 fold expansion, >93% purity, >98% viability, and stable CAR expression. At an effector-to-target ratio of 0.5:1, CD33-CD73 CAR-NK cells mediated ~80% specific lysis, with superior cytotoxicity vs conventional CD33 CAR-NK cells. In xenografts, CD33-CD73 CAR-NK cells achieved robust tumor clearance, extended median survival by 24.5 days (59.5 vs 35 days) versus standard CD33 CAR-NK cells, and five out of six mice achieved long-term survival (>50 days). CONCLUSION: The CD33-CD73 dual-targeting CAR-NK platform synergistically targets AML cells and the adenosine-rich TME, exhibiting superior anti-leukemia efficacy. This strategy advances AML immunotherapy and provides a translational blueprint for TME-targeted therapies in other cancers.