How do we manufacture clinical-grade interleukin-15-stimulated natural killer cell products for cancer treatment?

BACKGROUNDCancer immunotherapy involving natural killer (NK) cells has gained interest. Here we report two methods to obtain interleukin (IL)-15-activated NK cells for clinical use. STUDY DESIGN AND METHODSIL-15-activated NK cell products were obtained after 1) enrichment from healthy haploidentical donors' peripheral blood mononuclear cells (PBMNCs) collected by nonmobilized apheresis by a two-step magnetic procedure, depletion of CD3+ cells followed by selection of CD56+ cells and ex vivo overnight stimulation with IL-15 (NKIL15); and 2) expansion using the K562-mb15-41BBL cell line (NKAE), from autologous PBMNCs from patients with multiple myeloma or expansion from healthy haploidentical PBMNCs obtained from whole blood using the same previous cell line. We analyzed the NK cell recovery and expansion, Tcell depletion, phenotype, cytotoxicity, safety, and genomic stability of two good manufacturing practices (GMP)-grade IL-15-activated NK cell products. RESULTSThe number of NK cells obtained from NKIL15 cell and NKAE cell products was similar; however, there were significantly fewer T cells in the NKIL15 cell product. The haploidentical NKAE cell product contained more T cells than the autologous NKAE cell product. The surface expression of the activating receptors CD69, CD25, natural killer group-2 member D receptor, NKp44, NKp46, NKp30, and DNA accessory molecule 1 was upregulated in both NK cell products. NKIL15 cell and NKAE cell products had significantly higher lytic activity than unstimulated NK cells and showed no lytic activity against PBMNCs from healthy donors. No genetic alterations or potential oncogenic effects were found. CONCLUSIONDifferent GMP-grade procedures can be used to obtain large numbers of highly IL-15-activated NK cells with extremely low Tcell content for clinical use.