Abstract
Background aims. Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. Methods. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Results. Using this system we produced up to 19 × 109 functional NK cells from unseparated apheresis products, starting with 15 × 107 CD3– CD56 + NK cells, within 8–10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 + T cells within the NK cultures. However, these CD3 + T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. Conclusions. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.
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.