ABSTRACT
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 500 U/mL of rhIL2 (Proleukin), and 2.5% human serum/plasma to achieve concentrations sufficient to treat patients with >5210(10) cells. The protocol involves cultures initiated at 2.5210(5) 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 1.1-1.3210(6) cells/mL in a 1.2 L volume (1.36-1.56210(9) 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 trial 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.