Human natural killer cell manufacturing using a closed system process for patients with metastatic solid tumors or hematologic malignancies

Abstract

Background & Aim As potent effectors of the innate immune system natural killer (NK) cells are currently utilized in clinical trials world-wide to treat a variety of hematologic and solid organ malignancies. The ongoing phase I/II study at the National Institutes of Health (08-H-0186) previously utilized open-system processing requiring 60-80 small GREX flasks and single donor AB plasma in the culture medium. We optimized the manufacturing process to generate large numbers of NK cells using closed-system processing with large 5 liter GREX containers and commercially available pooled AB serum. Methods, Results & Conclusion The manufacturing process entails two stages: Preparation of the feeder EBV-transformed lymphoblastoid cell line (LCL) and NK cell culture. LCL are cultured in closed GREX100-CS vessels with XVIVO15 culture media containing 15mM HEPES, 2mM glutamax and 10% of either commercially available pooled AB serum or single donor AB plasma. With one culture vessel capable of generating 8-10 × 108 LCL feeder cells using 600-800mL of culture medium, 3 vessels sufficiently support a full scale culture. LCL number and viability are similar between groups containing serum vs. plasma. In the second stage, NK cell culture is initiated using 3 GREX500-CS with 40 × 106 NK cells and 400 × 106 irradiated LCL feeder cells in 500mL/vessel. Fresh XVIVO20 containing 15mM HEPES, 2mM glutamax, 500IU/mL IL2 and either 10% AB plasma or pooled AB serum is added on days 4, 8, 12 and 17. On day 14, a portion of the cells are harvested and washed for fresh infusion, and the remaining cells are re-seeded into a new vessel for harvest for second infusion on day 19. Day 14 and day 19 harvest generated 10-14 × 109 (1 vessel) and 50-70 × 109 NK cells (4-5 vessels) for fresh infusion, enough to support the target dose level of 1 × 108 (1st harvest) and 5 × 108 (2nd harvest) NK cells/kg. We found no significant differences in cell growth, viability or TRAIL expression when using AB plasma vs. AB serum. However, TRAIL expression was significantly increased when the concentration of the NK cells was kept below 4 × 106 cells/mL during culture. NK cells also demonstrated tumor cell line killing ability in vitro and had increased CD107. In summary, we have developed a new process that improves safety and consistency for culturing NK cells in large numbers for an ongoing clinical trial at the NIH. This process can be easily adapted by cell therapy manufacturing facilities at most academic institutions.