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Abstract
Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy.
Highlights
Natural Killer (NK) cells are CD56+CD32 large granular lymphocytes that exert innate immunity against viral infections and cancer [1]
We observed after 3 weeks total culture duration that the percentage of CD34+ cells further declined, while the CD56+CD161+CD94+ NK cell population increased to 10–18% (e.g. Figure 2a)
Our method enables the generation of functional human NK cells more than 4-logs from CD34+ cells enriched from freshly collected umbilical cord blood (UCB) units and more than 3-log from frozen UCB
Summary
Natural Killer (NK) cells are CD56+CD32 large granular lymphocytes that exert innate immunity against viral infections and cancer [1]. Ruggeri et al demonstrated that NK cell alloreactivity can control relapse of acute myeloid leukemia (AML) without causing graft-versus-host disease (GVHD) in the setting of HLA-mismatched haploidentical allogeneic SCT [4]. Haploidentical NK cell infusions together with IL-2 in a non-transplantation setting have been associated with complete hematologic remission in poor-prognosis patients with AML [5]. These encouraging results point out that allogeneic NK cell-based immunotherapy may be a promising therapeutic strategy for AML in both the non-transplant and posttransplant setting [5,6]
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