Natural killer (NK) cell-based adoptive immunotherapy is a promising treatment approach for many cancers. However, development of protocols that provide large numbers of functional NK cells produced under GMP conditions are required to facilitate clinical studies. In this study, we translated our cytokine-based culture protocol for ex vivo expansion of NK cells from umbilical cord blood (UCB) hematopoietic stem cells into a fully closed, large-scale, cell culture bioprocess. We optimized enrichment of CD34⁺ cells from cryopreserved UCB units using the CliniMACS system followed by efficient expansion for 14 days in gas-permeable cell culture bags. Thereafter, expanded CD34⁺ UCB cells could be reproducibly amplified and differentiated into CD56⁺CD3⁻ NK cell products using bioreactors with a mean expansion of more than 2,000 fold and a purity of >90%. Moreover, expansion in the bioreactor yielded a clinically relevant dose of NK cells (mean: 2×10⁹ NK cells), which display high expression of activating NK receptors and cytolytic activity against K562. Finally, we established a versatile closed washing procedure resulting in optimal reduction of medium, serum and cytokines used in the cell culture process without changes in phenotype and cytotoxic activity. These results demonstrate that large numbers of UCB stem cell-derived NK cell products for adoptive immunotherapy can be produced in closed, large-scale bioreactors for the use in clinical trials.

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units. Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 106/kg body weight) after lymphodepleting chemotherapy without cytokine boosting. Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1 × 104 T cells/kg and <3 × 105 B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly, in vivo HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (<0.1%), which lasted for 6 months. Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential “off-the-shelf” translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107–18. ©2017 AACR.

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.