IPSC-Derived CAR-NK Extracellular Vesicles Remodel Macrophage Immunity and Enhance Tumor Eradication

Chimeric antigen receptor (CAR) cell therapy has revolutionized the treatment of hematological malignancies; however, its efficacy in solid tumors remains limited due to dense stromal barriers and the immunosuppressive tumor microenvironment (TME), which hinder immune cell infiltration and persistence. To overcome these challenges, we propose an innovative strategy utilizing induced pluripotent stem cell (iPSC)-derived CAR-NK extracellular vesicles (CAR-iNEV), engineered from iPSC-differentiated NK cells. Unlike conventional CAR-NK cell therapies that rely on immortalized NK92 cell lines, our approach leverages iPSC-derived NK cells, reducing tumorigenic risk and enhancing CAR transduction efficiency. Additionally, we incorporate nanobody-based CAR constructs in place of traditional single-chain variable fragments (scFv), significantly improving CAR stability and expression efficiency. Beyond direct cytotoxicity, CAR-iNEV offers superior therapeutic advantages, including reduced systemic toxicity and enhanced drug delivery capabilities compared to CAR-iNK cells. In vivo studies demonstrate that CAR-iNEV exhibits exceptional safety and potent antitumor activity across multiple solid tumor xenograft and patient-derived xenograft (PDX) models in humanized mice. Mechanistically, CAR-iNEV not only directly eliminates tumor cells but also reprograms the TME by activating macrophage-derived nitric oxide synthase (NOS2), amplifying host antitumor immunity. These findings establish CAR-iNEV as a promising platform for solid tumor immunotherapy, particularly in relapsed and metastatic settings, providing a novel and effective strategy that bridges direct tumor targeting with immune microenvironment remodeling. This study lays the foundation for future clinical translation, advancing the next generation of cell-free CAR-based immunotherapies.