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 (refers as CIML NK, cytokine induced memory-like NK cells), demonstrating enhanced effector function and prolonged persistence. Preclinical studies and very early clinical trials revealed the promising clinical activity of CIML NK cells in the treatment of hematological malignancies, especially acute myelocytic leukemia (AML). However, the current manufacturing method for CIML NK cells do not endow these cells 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 (PBNK) cells by more than 200-fold within two weeks, with a purity exceeding 95%. Importantly, these ex vivo expanded NK (ExNK) cells exhibited hypomethylated state in the CNS-1 region (Conserved Noncoding Sequence 1) of IFNG gene, stronger metabolic ability, and enhanced effector function when compared to CIML NK cells. Based on these features, we named these expanded NK cells but still possess memory-like characteristics as mExNK (memory-like expanded NK cells). Bulk mRNA-seq further uncovered a high similarity between mExNK and CIML NK cells, with both highly expressing genes being related to proliferation, metabolism, and memory. Mouse tumor models proved that the infused mExNK exhibited longer persistence in vivo and stronger effector functions. Taken together, the method described herein showed an enhanced expansion efficiency for human CIML NK cells and would facilitate their clinic applications in cancer therapy.