Superior Expansion and Cytotoxicity of Human Primary NK and CAR-NK Cells from Various Sources via Enriched Metabolic Pathways

cytotoxicity from various sources (including peripheral blood, cord blood, and tumor tissue). We apply a human B-lymphoblastoid cell-line 721.221 (hereinafter, 221)-based artificial feeder cell system with membrane-bound interleukin 21 (mIL-21) to propagate NK and CAR-NK cells. The expansion capability, purity, and cytotoxicity of NK cells expanded with 221-mIL-21 feeder cells are superior to that of conventional K562-mIL-21 feeder cells. RNA sequencing (RNA-seq) data show that 221-mIL-21 feeder cell-expanded NK cells display a less differentiated, non-exhausted, limited fratricidal, memory-like phenotype correlated with enriched metabolic pathways, which explains underlying mechanisms. Thus, "off-the-shelf" NK and CAR-NK cells with superior functionalities and expansion using a genetically modified 221-mIL-21 feeder cell expansion system will greatly support clinical use of NK immunotherapy.