Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

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.

Expanded and Activated Natural Killer Cells for Immunotherapy of Hepatocellular Carcinoma

“…Based on our results, the antitumor potential of expanded NK cells, particularly when armed with NKG2D-CD3z-DAP10 receptors, should be much higher than that of cytokine-activated lymphocytes. Importantly, the method used in our study to expand NK cells is robust and has already been adapted to clinical-grade conditions (25,38).…”

“…The results of this study suggest that infusions of expanded NK cells with expression of NKG2D-CD3z-DAP10 receptors could be a useful addition to current treatment options, such as sorafenib and experimental therapies based on TCR-directed T cells. NK cells expanded with the method described in this study are being infused systemically in clinical trials for hematologic and nonhematologic malignancies (25,38). In HCC, intra-arterial delivery of expanded NKG2D-CD3z-DAP10 þ NK cells might also be explored (43).…”

Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein

“…[28][29][30] Several groups are actively investigating methods to expand NK cells ex vivo using genetically engineered antigen-expressing cells with membrane-bound IL-15, IL-21, and 41BB ligand expression to overcome the limitations of in vivo NK-cell expansion. [31][32][33] These NK cell products should be tested clinically against the fresh activated NK cell products described here.…”

T-cell and natural killer cell therapies for hematologic malignancies after hematopoietic stem cell transplantation: enhancing the graft-versus-leukemia effect

“…Although subsequent purification steps yielded higher purity, a substantially increased processing time (and consequent lower recovery) was needed. 87 Enrichment steps involve the cytokines IL-2, IL-15, or various feeder cells (K562 cells of myeloid lineage modified to express IL-15, 89 irradiated autologous peripheral blood mononuclear cells in the presence of IL-2, 90 or autologous mesenchymal stromal cells 91 ). Both IL-2 and IL-15 appear equally efficacious cytokines for expanding NK cells ex vivo and no added benefit was seen when both were combined or combined with other cytokines (e.g.…”