Abstract

Natural Killer (NK) cells show potent anti-leukemic activity in vitro and thus NK cells isolated using the CliniMACS® Cell Separation System are currently under clinical evaluation as part of a treatment for acute myeloid leukemia (AML). (Uharek L et al., 2003, Gentilini C et al., 2005; Koehl U et al., 2004, 2005; Passweg, JR et al., 2005, 2006; Miller J et al., 2005). Currently, the processing of highly purified, clinical-grade NK cells requires a two-step method using the CliniMACS Instrument: Depletion of CD3+ cells followed by enrichment of CD56+ cells (Iyengar R et al., 2003). This procedure shows excellent performance (reviewed in Passweg et al., 2005) with yields of 30–60%, purities of >85%, > 99.97%/3.5 log T cell depletion but is time consuming. Simpler one-step procedures have thus been tried to reduce the processing time (CD3 depletion, CD3/CD19 depletion, CD6 depletion, CD56 enrichment) but result in a more heterogeneous final cell product that usually contains less than 60% NK cells. NKp46 (CD335) has been demonstrated to be primarily expressed by NK cells and may thus be a suitable selection marker for one-step NK cell enrichment. We evaluated an NKp46 enrichment strategy using the autoMACS™ Pro Separator, NKp46-Biotin, and Anti-Biotin MicroBeads. We could enrich CD56+CD3- cells from peripheral blood mononuclear cells (PBMCs) to a purity of 90% with a yield of 40–60% and a >99.92%/3.1 log depletion of T cells (n = 2). These results encouraged the evaluation of this procedure on a clinical scale by using the CliniMACS Cell Separation System. Using the CliniMACS Plus Instrument, NK cells could be enriched to 73–87% purity (n = 3) from Buffy Coats derived from whole blood. T cells were efficiently depleted (>99.92%/3.0 log) to such a degree that NKp46-selected NK cell products may fulfil the release criteria for NK cell donor lymphocyte infusions (NK-DLI) in the context of haploidentical stem cell transplantation. While CD56+ CD3- NK cells are not activated by antibody binding during cell separation, magnetic labelling using anti-NKp46 antibodies may trigger NK cell activation. NKp46-mediated NK cell activation may be synergistically enhanced by the use of IL-2. Functional studies of NK cells isolated by different methods will be required to further evaluate their feasibility for use in NK-DLIs.

Author notes

Disclosure:Employment: All authors employed at Miltenyi Biotec GmbH.