Work

30: Ex-vivo Expansion and mRNA Transfection of Cord Blood Derived Natural Killer Cells

Year: 2007

Type: article

Abstract: Natural Killer (NK) cell-mediated alloreactivity 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 cell progenitors and represents an attractive platform for cytotoxic cell therapy. Our objective was to ex-vivo expand cord blood NK cells followed by genetic modification to ultimately render them cancer cell specific by introducing chimeric antigen receptors. CB mononuclear cells were cryopreserved after CD3 depletion performed by immunomagnetic microbead selection (Miltenyi Biotec, Auburn, CA). Cells were plated for NK expansion, with or without a feeder layer of irradiated umbilical cord mesenchymal cells (UC-MSC) obtained from the Wharton’s jelly 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 UC-MSC 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 UC-MSC feeder layers (29.6 ± 26.8 vs. 12.8 ± 8.9 fold, p=0.243). NK cells expansion was directly proportional to the number of UC-MSC plated as feeders. Expanded CB-NK cells also displayed enhanced cytotoxicity compared to control cultures 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). To test whether expanded CB-NK cells can be genetically engineered for ultimately targeting malignant cells, we transfected via electroporation (Bio-Rad) expanded CB-NK cells with mRNA produced from plasmid GFP-cDNA. GFP-mRNA expression (flow cytometry) at 24 hours was significantly higher (mean 42.8±5.2%) compared to 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 on a feeder layer of UC-MSC while preserving cytotoxicity and can also be genetically engineered by mRNA transfection.

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.bbmt.2007.08.046