Abstract 883: Scalable differentiation of cytotoxic natural killer (NK) cells from human pluripotent stem cells

Cell therapy is expanding as a treatment for various cancers. FDA-approved autologous therapies, such as chimeric antigen receptor (CAR) T cells and tumor infiltrating lymphocytes (TILs), are used for aggressive or late-stage cancers. However, these therapies face challenges due to lengthy manufacturing times and patient variability. Manufacturing is already a bottleneck, which is expected to worsen as these therapies are tested in earlier cancer stages and become available to more patients. An emerging solution for cancer treatment is the use of natural killer (NK) cells as an "off-the-shelf" therapy. These cells target malignant cells independent of antigens, without needing HLA matching, and have not caused cytokine release syndrome like some T cell therapies. Clinical trials typically administer 5x10^6 to 1x10^8 NK cells per kilogram of body weight per dose. Therefore, developing methods to produce large quantities of functional NK cells is crucial. Current methods, such as sourcing primary cells or differentiating from human pluripotent stem cells (PSCs) in two dimensions (2D), yield variable results, often require co-culture or embryoid body formation, and usually fail to produce enough functional NK cells for therapy. To address these limitations, we developed a scalable 3D PSC culture differentiation protocol to hematopoietic stem cells (iHSCs), a multipotent progenitor cell population, and then to induced natural killer (iNK) cells that primarily uses GMP grade reagents with regulatory documentation. Differentiation occurs in suspension using shake flasks. After 38 days, over 50% of cells are CD56+ CD3-, and can be cryopreserved, thawed, and further expanded. This method, tested in up to 100 mL cultures, produces 5-10x106 CD56+CD3- iNK cells/mL, sufficient to treat one patient at the lower dosing range. Multiple 100 mL cultures can be cultivated in parallel to increase the yield, and future efforts will focus on optimizing the protocol in larger culture volumes. To evaluate iNK cell cytotoxicity, they were co-cultured with three-dimensional (3D) patient-derived cancer organoids, a physiologically relevant in vitro cancer culture that better mimics tumor tissue than traditional 2D cancer cell lines and are harder to kill. iNK cells induced apoptosis in breast, colorectal, and endometrial cancer organoids in a dose-dependent manner. Against the same target colorectal cancer organoids, iNKs were more cytotoxic than the NK-92 cell line at equal doses, and matched primary NK cells at roughly double the dose. In summary, we demonstrate a novel scalable protocol for generating functional iNK cells, showing potency in a cytotoxicity assay with patient-derived cancer organoids, which could be further developed for cell therapy research. Citation Format: Marcus Bunn, Lindsay Bailey Steinitz, Vivek Chandra, Michael Akenhead, Logan Wilson, Erica Heipertz, Chris Yankaskas, Colin Paul, Matt Dallas, Mark Kennedy, David Kuninger. Scalable differentiation of cytotoxic natural killer (NK) cells from human pluripotent stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 883.