210 A novel medium for increased cytotoxicity and serum-minimal ex vivo expansion of natural killer (NK) cellular immunotherapies

BackgroundDespite significant clinical breakthroughs,1 2 cellular immunotherapy remains unacceptably variable in performance3 and impractically expensive for mainstream adoption.4 These hurdles are particularly true for new cancer immunotherapies with natural killer (NK) cells. Culturing NK cells ex vivo is challenging due to short half-lives, reduced functionality, and increased exhaustion.5 Also, the use of serum introduces inconsistency and increases cost.6 Here we introduce a novel prototype medium specifically formulated for NK cells, which uses a unique combination of plant extracts and molecular ingredients to increase cytotoxic performance while allowing the reduction of serum by up to 90 percent.MethodsKHYG-1 NK cell line, or primary NK cells, were brought to 2% or 2.5% serum, respectively, in the prototype media. DMEM/F12 with 20% serum (for KHYG-1) or 10% serum (for primary cells) was the control. All media was supplemented with 100 U/mL IL-2. Cell numbers were periodically assessed, using a cell counter or flow cytometry. For cytotoxicity assays, K562 target cells were cocultured with KHYG-1 cells (effector:target ratio 20:1) for 5h, or with primary NK cells (4:1) for 18h. Cells were stained with annexin V and propidium iodide to determine the levels of apoptosis and necrosis by flow cytometry. Levels of secreted proteins were determined using Luminex. Expression of CD56 was determined by flow cytometry.ResultsFollowing adaptation to 2% serum, growth of KHYG-1 cells in the prototype media stayed nearly on pace with the control for over 3 weeks. Human primary NK cells grew 10-fold over 10 days in the prototype media with 2.5% serum, without feeder cells; in contrast, primary cells did not proliferate in control medium (figure 1). Beta testing confirmed that the prototype media improved the proliferation of primary NK cells over a 12-day period, relative to a gold standard medium (figure 2). After culture in prototype media, both KHYG-1 cells and primary NK cells exhibited higher cytotoxic activity toward K562 cells, compared with the activity of the same cells cultured in control medium (figure 3). Secretion of interferon-gamma, perforin, and granzyme A was increased in KHYG-1 cells cultured in prototype media (figure 4). Additionally, culture in this media pushes NK cells toward a more CD56-dim phenotype (figure 5), which is associated with increased cytotoxic activity.Abstract 210 Figure 1Growth of NK cell line and human primary NK cells. (A)