Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children

Background: Allogeneic natural killer (NK) cells are known to show medium to high cytotoxic activity against HLA-nonidentical leukemia or tumor cells. For a possible benefit of postransplant treatment with NK cells after haploidentical stem cell transplantation (haplo-SCT) we developed a clinical scale procedure for NK cell processing observing Good Manufacturing Practice (GMP). Methods: Allogeneic donor Nl(cells were selected from 15 unstimulated leukaphereses using two rounds of immunomagnetic T cell depletion, followed by an NK cell enrichment step. CD56(+)CD3(+) NK cells were stimulated and expanded in vitro according to GMP. Quality control of NK cell purity, residual Tcells and cytotoxic activity was done by multi-coloured flow-cytometric analyses. Results: Purification led to an absolute number of 234-1237 x 10(6) CD56(+)CD3(-) NK cells from leukapheresis harvests with a median purity of 95% and a 4 to 61/2 log depletion of Tcells. After two weeks stimulation with IL-2 a fivefold expansion of Nl(cells with a Tcell contamination below 0.1% was reached. Median cell viability was 95 % after purification and 99% after expansion. The IL-2 stimulated Nl(cells showed a highly increased lytic activity against the MHC-I deficient K562 cells compared to freshly isolated NK cells and a medium cytotoxicity against patients' leukemic cells. Conclusions: Clinical scale enrichment and activation of allogeneic donor NK cells is feasible. High dose NK cell application may be a new treatment option for pediatric patients with leukemia or solid tumors in case of minimal residual disease or inbalanced chimerism post haplo-SCT as we could show for the first three patients [12].