Ex-Vivo Expansion and mRNA Transfection of Cord Blood Derived Natural Killer Cells with Preserved Cytotoxicity.

Natural Killer (NK) cell-mediated cytotoxicity can control leukemia relapse while protecting patients from graft-versus-host disease (GVHD) after allogeneic stem cell transplant. Cord blood (CB) is rich in NK cells with similar properties of proliferation and cytotoxicity as adult blood NK cells. Hence these cells are attractive for developing strategies to eliminate residual disease after cord blood transplant. In this study, CB mononuclear cells were cryopreserved after CD3 depletion performed by immunomagnetic microbead selection (Miltenyi Biotec, Auburn, CA). After thawing, cells were plated for NK expansion, with or without a feeder layer of irradiated cord mesenchymal (UCM) cells either from the same (autologous) or from an unrelated (allogeneic) cord donor, with or without IL-2 (1000 IU/ml), IL-15 (10 ng/ml), IL-3 (10ng/ml) and Flt3 (10ng/ml). At a median of 19 days of culture (range 14–21), there was significantly higher expansion (range 3.5–72 fold) of CD56⁺/CD3⁻ cells with the UCM feeder layer and cytokines compared to controls (mean 21.2 ± 20.8 fold increase vs 1.6 ± 0.9 fold increase with feeder layer only and 1.8 ± 0.89 fold increase with cytokines only, p=0.039 and p=0.041 respectively). There was no significant difference in NK expansion between autologous and allogeneic UCM feeder layers (29.6 ± 26.8 vs. 12.8 ± 8.9 fold, p=0.243). Expanded CB-NK cells were then tested for cytotoxicity against K562 cells using a calorimetric assay with PKH67-GL. CB-NK cells expanded either with autologous or allogeneic UCM feeder layers displayed enhanced cytotoxicity compared to controls plated with cytokines only (91.78±0.7% vs. 82.5±1.8%, p=0.003 and 89±2.3% vs. 83.7±0.18%, p=0.056, respectively). In order to test whether expanded CB-NK cells can be transfected for ultimately targeting malignant cells, we electroporated expanded CB-NK cells with mRNA produced from plasmid GFP-DNA by in vitro transcription. Flow cytometry was used to detect viability, which was 94%, 92% and 93% for non-transfected, GFP-DNA and GFP-mRNA samples respectively. GFP-mRNA expression at 24 hours was significantly higher (range 36.6–50.8%, mean 42.8±5.2%) compared GFP-cDNA controls (mean 4.2±0.35%, p<0.001). Mean GFP-mRNA expression was 35%, 31% and 16.5% at 48, 72 and 144 hours respectively. In summary, CB-NK cells can be effectively expanded with a feeder layer of UCM cells while preserving cytotoxicity and can also be genetically modified by mRNA transfection.