Cytokine-induced expandable memory NK cells with significant metabolic, epigenetic remodeling and persistence

Recent investigations have indicated that NK cells induced overnight with IL-12/15/18 cytokines possess memory-like characteristics, demonstrating enhanced effector function and prolonged persistence. Preclinical studies and very early clinical trials revealed the promising clinical activity of these cells in the treatment of hematological malignancies, especially acute myelocytic leukemia (AML). However, the current manufacturing method for these cells does not endow them with the ability to expand robustly ex vivo. The low production efficiency limits their clinical application. In this study, we established a novel expansion platform for generating memory-like NK cells by first inducing memory properties through stimulation with CD16a, IL-15, and IL-18, followed by a robust expansion phase. This method can effectively and stably expand healthy donor-derived Peripheral NK cells by more than 200-fold within two weeks, with a purity exceeding 95%. Importantly, these ex vivo expanded NK cells exhibited a hypomethylated state in the CNS-1 region of the IFNG gene, stronger metabolic ability, and enhanced effector function when compared to the original cytokine-induced memory-like NK cells. Based on these features, we named these expanded NK cells as memory-like expanded NK cells. Bulk mRNA-seq further uncovered a high similarity between these cells and the original cytokine-induced memory-like NK cells, with both highly expressing genes related to proliferation, metabolism, and memory. Mouse tumor models proved that the infused cells exhibited longer persistence in vivo and stronger effector functions. Taken together, the method described herein showed an enhanced expansion efficiency for human cytokine-induced memory-like NK cells and would facilitate their clinical applications in cancer therapy.