Cultivating Functional Natural Killer Cells from Mobilized Hematopoietic Stem Cells in Heavily Pretreated Hematologic Malignancies

CD19 chimeric antigen receptor (CAR) T cells have demonstrated promising outcomes in B-cell malignancies. However, using pretreated autologous T cells currently faces limitations, including compromised T-cell fitness and the challenge of manufacturing sufficient cell numbers for treatment. Consequently, natural killer (NK) cells have emerged as an alternative due to their natural ability to mediate cytotoxicity and their favorable safety profile. This study aims to generate patient autologous hematopoietic stem cell-derived NK (HSC-NK) cells and assess their therapeutic potential compared to peripheral blood NK (PB-NK) cells. We successfully cultivated HSC-NK under a 28-day, two-step differentiation and expansion protocol, achieving a cumulative 290-fold expansion using optimized memory-like cytokines and feeder cell stimulation. The expanded HSC-NK cells demonstrated a distinct phenotype (CD56+CD16low), representing an immature differentiation state, characterized by a lower expression of inhibitory receptors (NKG2A, KIR2DL, and CD94) and the exhaustion markers (LAG3, PD-1, TIM-3, and CTLA-4) compared to PB-NK cells. Prominent expression of CD62L, alongside sustained expression of CD69 and CD107a, was observed, translating into NK cell proliferation, activation, and cytotoxicity against cancer cells comparable to PB-NK cells. In conclusion, generating HSC-NKs is feasible while preserving essential NK cell phenotypes and activities. Our findings emphasize the potential of HSCs as an alternative NK cell source for cancer immunotherapy.