An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood

Tsutomu Nakazawa; Ryosuke Maeoka; Takayuki Morimoto; Ryosuke Matsuda; Mitsutoshi Nakamura; Fumihiko Nishimura; Shuichi Yamada; Ichiro Nakagawa; Young-Soo Park; Toshihiro Ito; Hiroyuki Nakase; Takahiro Tsujimura

doi:10.1016/j.reth.2023.05.006

An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood

Regen Ther. 2023 Jun 1:24:32-42. doi: 10.1016/j.reth.2023.05.006. eCollection 2023 Dec.

Authors

Affiliations

¹ Grandsoul Research Institute for Immunology, Inc., Uda, Nara, 633-2221, Japan.
² Clinic Grandsoul Nara, Matsui 8-1, Uda, Nara, 633-2221, Japan.
³ Department of Neurosurgery, Nara Medical University, Kashihara, Nara, 634-8522, Japan.
⁴ Department of Immunology, Nara Medical University, Kashihara, Nara, 634-8522, Japan.

Abstract

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, FcγRIII 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.

Keywords: Allogeneic NKC; Cancer immunotherapy; Cell-based immunotherapy; Cord blood; Glioblastoma.