An Efficient Feeder-Free and Chemically-Defined Expansion Strategy for Highly Purified Natural Killer Cells Derived from Human Cord Blood

Introduction: Natural killer cells (NKCs) are immune cells that can attack cancer cells through the direct recognition of ligands without prior sensitization. Cord blood-derived NKCs (CBNKCs) represent a promising tool for allogenic NKC-based cancer immunotherapy. Efficient NKC expansion and decreased T cell inclusion are crucial for the success of allogeneic NKC-based immunotherapy without inducing graft -versus-host reactions. We previously established an efficient ex vivo expansion system consisting of highly purified-NKCs derived from human peripheral blood. Herein, we evaluated the performance of the NKC expansion system using CB and characterized the expanded populations.Methods: Frozen CB mononuclear cells (CBMCs), with T cells removed, were cultured with recombinant human interleukin (rhIL)-18 and rhIL-2 under conditions where anti-NKp46 and anti-CD16 antibodies were immobilized. Following 7, 14, and 21 days of expansion, the purity, fold-expansion rates of NKCs, and the expression levels of NK activating and inhibitory receptors were assessed. The ability of these NKCs to inhibit the growth of T98G, a glioblastoma (GBM) cell line sensitive to NK activity, was also examined.Results: All expanded T cell-depleted CBMCs were included in over 80%, 98%, and 99% of CD3-CD56+ NKCs at 7,14, and 21 days of expansion, respectively. The NK activating receptors LFA-1, NKG2D, DNAM-1, NKp30, NKp44, NKp46, FcgRIII and NK inhibitory receptors TIM-3, TIGIT, TACTILE, NKG2A were expressed on the expanded-CBNKCs. Two out of three of the expanded-CBNKCs weakly expressed PD-1, yet gradually expressed PD-1 according to expansion period. One of the three expanded CBNKCs almost lacked PD-1 expression during the expansion period. LAG-3 expression was variable among donors, and no consistent changes were identified during the expansion period. All of the expanded CBNKCs elicited distinct cytotoxicity-mediated growth inhibition on T98G cells. The level of cytotoxicity was gradually decreased based on the prolonged expansion period.Conclusions: Our established feeder-free expansion system yielded large scale highly purified and cytotoxic NKCs derived from human CB. The system provides a stable supply of clinical grade off-the -shelf NKCs and may be feasible for allogeneic NKC-based immunotherapy for cancers, including GBM.& COPY; 2023, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).