Background The use of allogeneic natural killer (NK) cell as an adoptive immunotherapeutic is a promising strategy for a large pool of cancer patients, ensuring greater time- and cost-efficiency than autologous NK or T cell-based immunotherapies. However, large-scale production of clinical-grade NK cells is challenging with often use of genetically modified feeder cells, and the impairment of cytotoxic function in vivo due to exhaustion or senescence is a significant concern.

Methods We produced highly potent NK cells called Tumor-targeting Optimally Primed NK cell (T.O.P. NK), which are CD3-CD56+ cells directly derived from human PBMCs. These cells are expanded in a 50L bioreactor without feeder cells or genetic modification.

Results The T.O.P. NK demonstrates an increased expression of chemokine receptors, CCR5 and CXCR4, and NK activation markers, such as NKG2D, NKp46, and DNAM-1; the NK inhibitory and exhaustion markers such as CD57, PD-1, and TIM-3 maintained low expression levels after the harvest. The elevated expression of CCR5 and CXCR4 on T.O.P. NK elucidates its active migration in response to CCL5/RANTES and CXCL12/SDF-1 ligands, respectively, with high sensitivity. Furthermore, T.O.P. NK showed robust cytotoxicity in vitro and in vivo against various solid cancer cell lines even after recovery from cryopreservation. We observed strong anti-tumor activity of T.O.P. NK in short-term and long-term in vitro killing assays against solid cancer cell lines, including colorectal, breast, and head and neck cancers. Moreover, T.O.P. NK elicited strong anti-tumor effects in immunodeficient NOG mouse grafted with various human cancer cell lines.

Conclusions Collectively, we report T.O.P. NK, which are generated at clinical grade, with high productivity and purity while maintaining active phenotype and anti-tumor cytotoxicity. These findings suggest that the feeder-free expansion platform of T.O.P. NK proficiently generates allogeneic adoptive NK cells for the treatment of solid cancers.

Ethics Approval The study was approved by EWHA Womans University Medical Center‘s Ethics Board, approval number SEUMC 2021-04-026