Cytokine pre-activation enhances PM21-particle driven NK cell expansion

Abstract

Background & Aim Natural Killer (NK) cells are innate cytotoxic lymphocytes with high potential for development of cell-based anti-cancer therapeutics. To be applied clinically, NK cells need to be expanded to achieve sufficient amounts for therapeutically effective dosages. Methods utilizing stimulation with membrane bound IL21 (mbIL21) expressed on K562 feeder cells (K562-mbIL21 cells) or on plasma membrane particles (PM21-particles) derived from those cells result in robust NK cell expansion sufficient to support such dosing. Furthermore, expanded NK cells are much more cytotoxic against various cancer cells, secrete large amounts of IFNγ and TNFα in response to cytokine and/or target cell stimulation, and have shown promising results in clinical trials for AML. Another method that has been applied clinically utilizes short overnight pre-activation with cytokines IL12/IL15/IL18 to induce memory-like NK cells with greater proliferative potential that can allow for enhanced in vivo NK cell expansion post-infusion. This study tested the hypothesis that cytokine pre-activation of NK cells prior to expansion with PM21-particle will result in enhanced NK cells expansion in vitro. Methods, Results & Conclusion NK cells from PBMCs obtained from healthy donors were pre-activated overnight with IL2; or IL12, IL15 and IL18. Unactivated PBMCs or pre-activated PBMCs were expanded by stimulating with PM21 particles (200 µg/mL) in SCGM media supplemented with 10% FBS and IL2 (100 U/mL). Cell content was tested every 2–3 days and media changed as needed. Cells expanded under different conditions were assayed for cytotoxicity and cytokine production in response to cytokines and/or tumor targets. Pre-activation of NK cells with cytokines IL12, IL18 and IL15 led to significantly improved NK cell expansion in response to PM21-particles (CAP, cytokine and particle) as compared to standard PM21-particle conditions (14,500 vs. 2,500, N=13, p=0.008-Figure 1). CAP-NK cells had comparably high cytotoxicity to PM21-NK cells. They also produced comparable level of IFNγ and TNFα in response to IL12/IL18 and/or K562 target cell stimulation. The new combined procedure with cytokine pre-activation and PM21-particle stimulation results in significantly improved NK cell expansion. The improved method allows for even greater manufacturing of therapeutic NK cells.