Seeding density is critical for optimizing scalable feeder-free natural killer cell expansion

Background & Aim Generating clinical doses of natural killer (NK) cells is an important challenge for Chimeric Antigen Receptor (CAR)-NK cell therapies. Current manufacturing methods to expand NK cells include irradiated feeder cells. However, these methods are restrictive due to high costs, scale-up difficulties, and licensing restrictions. In this study we investigated an optimized method for the expansion of NK cells in a feeder-free system that enables scalable cell expansion for NK therapies. Specifically, we investigated the impact of cell activation methods, cell culture vessels, & cell seeding density on NK expansion and purity. Methods, Results & Conclusion Human peripheral blood mononuclear cells (PBMCs) were cultured for 10-days in either G-Rex® 24-well plates, T25 Flasks, or G-Rex 6-well plates. PBMCs were analyzed using a flow cytometer for CD3−/CD56+ NK cells prior to expansion to determine NK seeding density. The Cloudz™ NK Cell Expansion Kit or Miltenyi MACS iBeads were used to activate & expand NK cells. A half-volume media change was performed every 3 days and cytokines were refreshed at the following concentrations: 27 ng/mL IL-2, and 10 ng/mL each of IL-12, IL-18, & IL-21. After 10 days in culture the cells were analyzed for CD3−/CD56+ NK cell expansion and purity, defined as % CD3−/CD56+ cells relative to the total population. We found that the Cloudz NK Cell Expansion Kit produced the greatest NK cell expansion & purity from PBMCs after 10 days of culture (318-fold NK cell expansion and 85% purity) when used with a cytokine combination of IL-2/IL-12/IL-18/IL-21. These data represent 6 experiments utilizing 85 samples across 5 separate donors. For comparison, MACS iBeads produced an average of 177-fold expansion and 64.5% purity. While developing a scalable protocol to allow flexibility in NK cell manufacturing, we found that cell seeding density was an important variable. In the 24-well G-Rex platform, a seeding density of 60,000 NK cells/cm2 yielded the highest growth and purity after 10-days. However, 30,000 NK cells/cm2 produced the highest growth rate in the 6-well G-Rex platform over 10 days (Figure 1). These results indicate that robust expansion & high purity can be achieved using feeder-free NK cell expansion methodologies. These data suggest that seeding density is a critical factor for cell expansion under feeder-free conditions and that utilizing the G-Rex platform can enable scalable expansion for cell therapy manufacturing.