Robust differentiation of NK cells from MSLN.CAR-IL-15–engineered human iPSCs with enhanced antitumor efficacy against solid tumors

Human induced pluripotent stem cells (iPSCs) offer a promising source for chimeric antigen receptor (CAR)-engineered natural killer (NK) products. However, complex iPSC-NK (iNK) manufacturing challenges clinical use. Here, we identified LiPSC-GR1.1 as a superior iPSC line for iNK production. By engineering LiPSC-GR1.1 with a mesothelin (MSLN)-targeting CAR and interleukin-15 (IL-15), we achieved robust differentiation of iPSCs into mature activated iNK cells with enhanced tumor killing efficacy, superior tumor homing, and vigorous proliferation. Single-cell transcriptomic analysis revealed that transforming growth factor-β (TGF-β)-producing tumor cells up-regulated major histocompatibility complex molecules and down-regulated MSLN post-CAR-IL-15 iNK treatment. Tumor-infiltrating CAR-IL-15 iNK cells exhibited high levels of CAR, IL-15, and NK-activating receptors, negligible checkpoint exhaustion markers, and extremely low levels of NK suppressive factors <i>CISH</i>, <i>TGFBR2</i>, and <i>BATF</i>, enabling them to sustain activation, metabolic fitness, and effective tumor killing within TGF-β-rich hypoxic tumor microenvironment. Overall, we developed MSLN.CAR-IL-15-engineered GR1.1-iNK therapy with enhanced antitumor efficacy for solid tumor treatment.