Robust differentiation of NK cells from MSLN.CAR-IL-15-engineered human iPSCs with enhanced anti-tumor efficacy against solid tumors [scRNA-seq]

Human induced pluripotent stem cells (iPSCs) offer a promising source for producing standardized, off-the-shelf CAR-NK products. However, the complex and time-consuming 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 IL-15, we achieved efficient robust differentiation of iPSCs into mature and activated iNKs, which demonstrated enhanced tumor-killing efficacy, superior tumor-homing, and vigorous proliferation. Single-cell transcriptomic analysis revealed that TGF-β-producing tumor cells upregulated MHC molecules and downregulated MSLN expression post-CAR-IL-15 iNK treatment. Tumor-infiltrating CAR-IL-15 iNKs 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 the TGF-β-rich hypoxic tumor microenvironment. Overall, we developed a MSLN.CAR-IL-15-engineered GR1.1-iNK product with enhanced anti-tumor efficacy for solid tumor treatment.