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 CISH, TGFBR2, and BATF, 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.