Abstract LB-072: Activating and expanding NK cells using dissolvable alginate particles functionalized with αNKp46 and αCD2
Authors: Johnson Christopher , Senior Hannah , Jesuraj Nithya 
Published: August 2019

Abstract
Introduction: Generating clinical scale doses of natural killer (NK) cells is currently a challenge for manufacturing CAR-NK cell therapies. Current methods to expand NK cells include irradiated feeder cells that have been engineered to mimic dendritic cells as well as commercially available combinations of antibody & proteins for targeted NK cell expansion. These methods are restrictive due to high costs, scale-up challenges, and licensing restrictions for clinical & commercial applications. We hypothesized that using a dissolvable phase-change hydrogel conjugated with ligands targeting NK cell activation may provide a more efficient and defined alternative for NK manufacturing processes. Additionally, we investigated cytokine culture combinations that optimize the expansion of NK cells.
Methods: Hydrogel microspheres (9 µm median diameter) - referred to as Cloudz™ particles - were functionalized with αNKp46 and αCD2 ligands using standard bioconjugation techniques. In this study, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days in G-Rex® 24-well plates containing NK cell-functionalized Cloudz™ particles along with various cytokine combinations. Prior to expansion, PBMCs were analyzed for CD3-/CD56+ NK cells and 6x105 NK cells were seeded into each well with a 2:1 ratio of Cloudz™ particles:NK cells. Miltenyi MACS iBeads were used as a control and were prepared according to the manufacturer instructions. During days 0-3, the cells were cultured with IL-2 (40 U/mL) along with combinations of IL-12, IL-18, and IL-21 (10 ng/mL each). After 3 days, the IL-2 concentration was increased to 200 U/mL, while the other cytokine concentrations remained constant. The cytokines and media were refreshed every 3 days with ½ media changes. After 7 days in culture the cells were analyzed by flow cytometry for CD3-/CD56+ NK cell expansion and purity. NK cell purity was defined as the percentage of CD3-CD56+ cells relative to the total population.
Results: Cultures using NK cell-functionalized Cloudz™ particles combined with IL-2 resulted in a 69% NK cell purity and 29.9-fold expansion. In comparison, cultures using MACS iBeads combined with IL-2 resulted in 49% NK cell purity and a 6.8-fold expansion, suggesting that Cloudz™-based methods provide better expansion. When IL-12 was added to cultures with Cloudz™ particles, both the purity and expansion decreased (45% and 7.2-fold, respectively). When IL-12 and IL-18 were added, the expansion increased to 33.9-fold and the purity increased to 60%. The best combination was IL-2/IL-12/IL-18/IL-21 which had an 88.9-fold expansion with a 78% purity. IL-12 and IL-18 mimic the activating signals released by dendritic cells and IL-2 and IL-21 mimic the activating signals released by activated T cells.
Conclusion: These data demonstrate that Cloudz™ particles functionalized with αNKp46 and αCD2 can efficiently induce NK activation and expansion with high purity and growth. In future studies, we will determine if NK cells expanded using this method show killing ability of cancer cells and persistence in in vivo models. We will also further explore the possibilities of this novel system for rapid expansion and recovery of specific NK cell subtypes.
Citation Format: Christopher Johnson, Hannah Senior, Nithya Jesuraj. Activating and expanding NK cells using dissolvable alginate particles functionalized with αNKp46 and αCD2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-072.