Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications
Introduction
Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that have direct and indirect roles in controlling intracellular pathogens. NK cells also play a major role in tumor immunosurveillance, as NK activity has been inversely correlated with cancer incidence and outcome (1., 2., 3., 4.) and NK infiltration of tumors is associated with better prognosis of gastric (5), colorectal (6) and lung carcinomas (7,8). Both primary NK cells derived from apheresis products and a pure NK cell line (irradiated NK-92) have been used as immunotherapy for blood and solid malignancies (9., 10., 11.). These studies have shown that NK cell infusions are well-tolerated, do not cause graft-versus-hostdisease (GvHD) or autoimmunity, and are associated with complete remission in poor-prognosis patients (9,10).
A major hurdle in NK cell clinical trials has been obtaining large numbers of NK cells with high purity and potency. Several protocols have been developed for ex vivo NK cell expansion using a range of cytokines, such as interleukin (IL)-2, IL-12 and IL-15, and feeder cells, including B-lymphoblastoid cell lines and monocytes (12., 13., 14., 15., 16.). Recently, a novel method of NK cell expansion using HLA-negative K562 cells genetically modified to express membrane-bound IL-15 and 4-1 BB Ligand (BBL), which specifically activate NK cells and promote their proliferation and survival, was reported (17,18). This strategy induced a median 21.6-fold expansion of NK cells in small-scale and 90.5-fold expansion in large-scale 7-day cultures (18). Despite progress made in ex vivo expansion of NK cells from peripheral blood precursors, manufacturing large numbers of pure NK cells for clinical trials requiring high infusion doses remains challenging. As a Center for Production Assistance for Cellular Therapies (PACT), NHLBI, we were charged with the manufacture of NK cells for the treatment of multiple myeloma (MM) for investigators at the University of Arkansas for Medical Sciences (Little Rock, AR, USA). The clinical protocol for this trial required up to 5 × 107 NK cells/kg and a CD3 depletion step (for allogeneic products), therefore we had to validate the manufacture of up to 10 × 109 total cells. These numbers would require cultures in more than 40 200-mL gas-permeable tissue culture bags with frequent feeding. We had recently evaluated gas- permeable cell culture devices (G-Rex) for the expansion of T cells and tumor cell lines, in which gas exchange across the base of the culture allows increased volumes of medium per unit area, increases the rate of cell expansion, decreases cell death and minimizes cell manipulation. We therefore evaluated NK cell expansion in the G-Rex and compared the process with that in the bags. The G-Rex supported more than 100-fold NK cell expansion within 8–10 days of culture without medium exchange. These cells had an activated NK cell phenotype and killed tumor cell targets, and retained viability and recovery after cryopreservation over a 12-month period.
Section snippets
Cells
Peripheral blood mononuclear cells (PBMC) were purified on Ficoll gradients from leukopacks (Gulf Coast Blood Center, Houston, TX) or apheresis products from consenting healthy volunteers and patients at the University of Arkansas for Medical Sciences. K562-mb15-41BBL was obtained from St Jude Children's Research Hospital (Memphis, TN, USA) (17,18). A master cell bank of K562-mbIL15-41BBL feeder cells was manufactured and characterized as a part of a PACT project in the good manufacturing
Optimization of NK cell expansion in the G-Rex
The Wilson–Wolf (G-Rex) devices support the growth of many suspension cells with minimal manipulation (19,20). To determine whether NK cells could also be expanded to large numbers, we first sought the optimal cell NK seeding density allowing us to grow these cells in G-Rex100 flasks containing 400 mL medium (Figure 1). The frequency of CD56 + CD3− NK cells in PBMC was determined by flow cytometry (Mean 8.8 ± 2.5%, n = 8; see Supplementary Figure 2 to be found online at //www.informahealthcare.com/doi/abs/10.3109/14653249.2012.700767
Discussion
We report an efficient and reproducible cell culture protocol for ex vivo expansion of activated NK cells in gas-permeable cell culture devices (G-Rex). This protocol is based on a previously described method of ex vivo NK generation from peripheral blood cells stimulated with genetically modified K562-mb15-41BBL cells, expressing membrane-bound IL-15 and 4-1BBL (17,18). The original protocol used gas-permeable bags for growing NK cells and frequent medium supplementation during culture. Here
Acknowledgments
This work was supported in part by NIH-NHLBI-N01 HB37163, NHLBI-1U54 HL081007 and NIH-NCI PO1 CA94234. We would like to thank Oumar Diouf, Deborah Lyon, Jeannette Bloom and Huimin Zhang and all the GMP staff of CAGT for the technical support in enabling this work.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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