Establishment of an efficient ex vivo expansion strategy for human natural killer cells stimulated by defined cytokine cocktail and antibodies against natural killer cell activating receptors

Abstract

Introduction: Cell-based immunotherapy is categorized as a regenerative therapy under the Regenerative Medicine Safety Act in Japan. Natural killer (NK) cell-based immunotherapy is considered a promising strategy for treating cancer, including glioblastoma (GBM). We previously reported an expansion method for highly purified human peripheral blood-derived NK cells using a cytokine cocktail. Here, we aimed to establish a more efficient NK cell expansion method as compared to our previously reported method.

Methods: T cell-depleted human peripheral blood mononuclear cells (PBMCs) were isolated from three healthy volunteers. The depleted PBMCs were cultured in the presence of recombinant human interleukin (rhIL)-18 and high-dose rhIL-2 in anti-NKp46 and/or anti-CD16 antibody immobilization settings. After 14 days of expansion, the purity and expansion ratio of CD3-CD56+ NK cells were determined. The cytotoxicity-mediated growth inhibition of T98G cells (an NK activity-sensitive GBM cell line) was evaluated using a non-labeling, impedance-based real-time cell analyzer.

Results: Anti-NKp46 stimulation increased the NK cell purity and expansion ratio as compared to the non-antibody-stimulated population. Anti-CD16 stimulation weakly enhanced the NK cell expansion ratio of the non-antibody-stimulated population and enhanced the NK cell purity and expansion ratio of anti-NKp46-stimulated populations. All NK cell-containing populations tested distinctly inhibited T98G cell growth. These effects tended to be enhanced in an NK cell purity-dependent manner. In some cases, anti-CD16 stimulation decreased growth inhibition of T98G cell compared to other conditions despite the comparable NK cell purity.

Conclusions: We established a robust large-scale feeder-free expansion system for highly purified human NK cells using a defined cytokine cocktail and anti-NK cell activating receptor antibodies. The expansion system could be feasible for autologous or allogeneic NK cell-based immunotherapy of GBM. Moreover, it is easily controlled under Japanese law on regenerative medicine.

Keywords: Cell-based immunotherapy; Glioblastoma; NK cells; PBMCs.

Fig. 1

Ex vivo expansion of human peripheral blood-derived NK cells by defined cytokine cocktail and NK cell activating receptor stimulation. Human CD3-depleted PBMCs were expanded with serum-free medium containing the defined cytokine cocktail and 10% autologous plasma on antibody immobilization plates. The dot plots depict the flow cytometric analysis data of CD3-and/or CD56-expressing cells. Day 0 indicates the CD3-depleted PBMCs. Day 14 indicates the 14-day expansion in the specific conditions. Ctr, non-immobilized; αCD16, anti-CD16 antibody; αNKp46, anti-NKp46 antibody; αCD16/αNKp46, anti-CD16/NKp46 antibody. Top, middle, and bottom panels indicate Donor 1, 2, and 3, respectively.

Fig. 2

Cytotoxicity-mediated growth inhibition of GBM cell by the expanded NK cell-containing populations. The NK cell-containing populations were co-cultured with attached T98G cells at an E:T cell ratio of 1:1. Left: Graphs depict representative data of real-time normalized cell index value. X-axis indicates time from target cell seeding. Y-axis indicates the normalized cell index calculated by the cell index of each point divided by the cell index of the co-culture start point. Right: Bar graphs illustrate the normalized cell index of each time point. Data are the mean ± SD of two independent experiments (n = 3–4). Statistical differences were determined by two-way ANOVA followed by Tukey's test. ∗∗p < 0.01, ∗p < 0.05, n.s.: not significant.