A Novel Method for Highly Efficient Ex Vivo Expansion and Genetic Engineering of Human Natural Killer Cells for Cancer Therapy

Cancer immunotherapy with adoptive transfer of human leukocyte antigen-mismatched, CD19-targetd chimeric antigen receptor (CAR)-transduced natural killer (NK) cells has attracted attention because of its efficacy and safety when infused in patients with refractory and relapsed B-cell lymphomas. However, generating clinical doses of CAR-NK cells is still a challenge. The methods for ex vivo expansion and genetic modification of primary human NK cells usually rely on the use of irradiated feeder cell lines, which has been restrictive due to high costs, scale-up difficulties, and licensing restrictions. Hence, novel strategies that do not require feeder cells will be beneficial in standardizing these types of cell therapies. In this study, we show the effectiveness of a novel feeder-free culture system in expanding NK cells ex vivo and generating CAR-NK cells. Unsorted peripheral blood mononuclear cells (PBMCs) collected from healthy donors were cultured with a reagent containing dissolvable microspheres that are conjugated with anti-CD2/NKp46 antibodies (Cloudz TM Human NK Cell Expansion Kit) and a combination of multiple cytokines, including interleukin (IL)-2, IL-12, IL-18, and IL-21 in medium supplemented with 10% fetal bovine serum. The activated NK cells were transduced using the RD114-pseudotyped retrovirus vector. To test whether the reagent promoted ex vivo NK cell expansion, we cultured PBMCs from 13 donors. The percentage of NK (CD56 + CD3 -) cells of initially isolated PBMCs was 15.3±7.5%. In the presence of multiple cytokine combinations, NK cell purity gradually increased and reached 91.6±7.6% by day 21. The NK cells expanded to 75.6±59.2-fold at day 10, 334±217-fold at day 14 and 1,542±913-fold at day 21. The expanded NK cells degranulated and produced intracellular cytokines upon exposure to K562 myeloid leukemia cells. The NK cells efficiently killed myeloid leukemia cells, such as K562, THP1, and KG1. The expression pattern of killer cell immunoglobulin-like receptors on NK cells remained unchanged. The expression of activating NK cell receptors, including NKp30 and NKp44, increased after 21 days of culture. Thereafter, a gene transfer to the primary human NK cells was conducted. We tested transduction efficiency and yields of modified cells on 7 days after the procedure by empty-vector transduction into NK cells expanded ex vivo for 3, 6, and 10 days (n=3, each). The results were presented as means ± standard deviation; 55.6±11.6%, 61.6±14.