Optimization of expansion techniques for adoptive NK cell transfer in dogs with cancer

Abstract Natural killer (NK) cells can recognize heterogeneous cancer cell targets without prior sensitization, making them promising prospects for use in immunotherapy. We have completed first-in-dog feasibility clinical trials in dogs with cancer using both autologous and allogeneic NK cells expanded from peripheral blood mononuclear cells (PBMCs). Previously, CD5 depletion of PBMCs has been used to enrich for a CD5dim expressing subset prior to NK co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. The purpose of this study was to compare ex vivo culture conditions using standard CD5 depletion versus unmanipulated PBMCs plus feeder line co-culture with 100 IU/mL rhIL-2 in matched healthy donors, hypothesizing that PBMCs plus feeder cells would generate an equivalent or superior NK product to CD5 depletion. Cell count, fold change, and viability data were collected for both techniques in 12 dogs across five time points up to day 14. A mixed-effects model analysis showed no statistical difference in calculated cell counts, overall fold change, and viability (p>0.05 all) between PBMCs with feeders and CD5 depleted cells with feeders. PBMCs had a higher mean than CD5 depleted cells at day 14 in all three categories, reaching a peak mean of 677 million cells from 5 million PBMCs at day 0. Flow phenotype showed similar upregulation of NKp46 and Granzyme B expression. Killing assays against melanoma and osteosarcoma targets demonstrated comparable results among PBMCs plus feeders versus CD5 depleted NK cells (p>0.05). Overall, these findings support the use of unmanipulated PBMCs plus feeder line coculture as an equivalent method to CD5 depletion in the expansion of canine NK cells for adoptive immunotherapy. Supported by grants from National Institutes of Health/National Cancer Institute (U01 CA224166-01, 1R03CA252793, T32 CA251007-01)