Ex vivo expansion of the highly cytotoxic human natural killer cell line NK-92 under current good manufacturing practice conditions for clinical adoptive cellular immunotherapy

Background Adoptive transfer of ex vivo expanded cytotoxic immune cells has become a viable strategy for treatment of malignant disease. Natural killer (NK)-92, a highly cytotoxic, IL2-dependent human NK cell-line, is an excellent candidate as an immunotherapeutic agent, being active for prolonged periods following irradiation and IL2 deprivation, non-toxic and non-immunogenic, and easily expanded. A number of clinical trials using NK-92 for different indications are currently underway. The aim of this study was to develop current good manufacturing practice (cGMP)-compliant expansion methodology for NK-92. Methods The ability to expand NK-92 ex vivo was evaluated. Serum-free culture media, as well as media supplements (IL2, serum/plasma/albumin), culture containers and feeding regimens were compared for their ability to support expansion, viability and cytotoxicity of NK-92 cells. Results NK-92 cells can be expanded in X-Vivo 10 serum-free media with 450 U/mL of rhIL2 (Proleukin), and 2.5% human serum/plasma to achieve concentrations sufficient to treat patients -with > 5 × 1010 cells. The protocol involves cultures initiated at 2.5 × 105 cells/mL in 25 mL in 1 L Vuelife culture bags, with addition of fresh media plus IL2 every 3 days to maintain an optimal density of NK-92 cells for expansion. Daily disruption of cell aggregates enhances NK-92 cells expansion and viability during the culture period. Final yields of approximately 1.1–1.3 × 106 cells/mL in a 1.2 L volume (1.36–1.56 × 109 cells; 218–250 fold expansion) over 15–17 days is achievable under cGMP-compliant conditions with > 85% viability. The feasibility of this approach has been shown in ongoing clinical trials with NK-92. Discussion We describe a protocol that allows for > 200-fold expansion of NK-92 cells within a 2–2.5 week period under GMP standards, in quality and quantity suitable for clinical adoptive immunotherapy. Adoptive transfer of ex vivo expanded cytotoxic immune cells has become a viable strategy for treatment of malignant disease. Natural killer (NK)-92, a highly cytotoxic, IL2-dependent human NK cell-line, is an excellent candidate as an immunotherapeutic agent, being active for prolonged periods following irradiation and IL2 deprivation, non-toxic and non-immunogenic, and easily expanded. A number of clinical trials using NK-92 for different indications are currently underway. The aim of this study was to develop current good manufacturing practice (cGMP)-compliant expansion methodology for NK-92. The ability to expand NK-92 ex vivo was evaluated. Serum-free culture media, as well as media supplements (IL2, serum/plasma/albumin), culture containers and feeding regimens were compared for their ability to support expansion, viability and cytotoxicity of NK-92 cells. NK-92 cells can be expanded in X-Vivo 10 serum-free media with 450 U/mL of rhIL2 (Proleukin), and 2.5% human serum/plasma to achieve concentrations sufficient to treat patients -with > 5 × 1010 cells. The protocol involves cultures initiated at 2.5 × 105 cells/mL in 25 mL in 1 L Vuelife culture bags, with addition of fresh media plus IL2 every 3 days to maintain an optimal density of NK-92 cells for expansion. Daily disruption of cell aggregates enhances NK-92 cells expansion and viability during the culture period. Final yields of approximately 1.1–1.3 × 106 cells/mL in a 1.2 L volume (1.36–1.56 × 109 cells; 218–250 fold expansion) over 15–17 days is achievable under cGMP-compliant conditions with > 85% viability. The feasibility of this approach has been shown in ongoing clinical trials with NK-92. We describe a protocol that allows for > 200-fold expansion of NK-92 cells within a 2–2.5 week period under GMP standards, in quality and quantity suitable for clinical adoptive immunotherapy.