Phenotypic and functional characterisation of locally produced natural killer cells ex vivo expanded with the K562-41BBL-mbIL21 cell line

We characterised the expansion, phenotype and functional activity of natural killer (NK) cells obtained for a clinical trial. Nineteen expansion procedures were performed to obtain NK cell products for 16 patients. NK cells were expanded ex vivo from haploidentical donor peripheral blood mononuclear cells in the presence of the locally generated feeder cell line K-562 with ectopic expression of 4-1BBL and mbIL-21. The median duration of expansion was 18 days (interquartile range 15-19). The median number of live cells yielded was 2.26 × 109 (range 1.6-3.4 × 109) with an NK content of 96.6% (range 95.1-97.9%). The median NK cell fold expansion was 171 (range 124-275). NK cell fold expansion depended on the number of seeded NK cells, the initial level of C-myc expression and the initial number of mature and immature NK cells. The majority of expanded NK cells had the phenotype of immature activated cells (NKG2A + , double bright CD56 + + CD16 + + , CD57-) expressing NKp30, NKp44, NKp46, NKG2D, CD69, HLA-DR and CD96. Despite the expression of exhaustion markers, expanded NK cells exhibited high cytolytic activity against leukaemia cell lines, high degranulation activity and cytokine production. There was a noted decrease in the functional activity of NK cells in tests against the patient's blasts.In conclusion, NK cells obtained by ex vivo expansion with locally generated K562-41BBL-mbIL21 cells had a relatively undifferentiated phenotype and enhanced cytolytic activity against cancer cell lines. Expansion of NK cells with feeder cells yielded a sufficient quantity of the NK cell product to reach high cell doses or increase the frequency of cell infusions for adoptive immunotherapy. Registered at clinicaltrials.gov as NCT04327037.