A good manufacturing practice method to ex vivo expand natural killer cells for clinical use

Abstract

Background: Great interest has been raised recently by the design of new adoptive immunotherapeutic strategies based on the in vivo infusion of ex vivo-expanded and activated natural killer (NK) cells. The development of good manufacturing practice (GMP) methods for the efficient production of fully functional NK cells is mandatory for clinical application.

Materials and methods: Peripheral blood mononuclear cells were obtained by leukapheresis and processed in the GMP facility. For NK-cell enrichment, a two-step immunomagnetic procedure consisting of CD3(+) T-cell depletion followed by CD56(+) cell positive selection was used. Isolated NK cells were suspended in serum-free medium containing autologous plasma, interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder cells and cultured for 14 days at 37 °C. IL-2 and IL-15 were also added during the last 24 hours of culture. Expanded cells underwent full quality control testing for cytogenetic characteristics, viability, sterility, phenotype and endotoxin status; functional tests, such as degranulation assays and cytotoxicity, were performed on expanded NK cells before cryopreservation and after thawing.

Results: NK-cell populations expanded on average 15.7±4.7 fold by day 14, with a viability of 96% ±0.5. At the end of the incubation period, 97% ±1.1 of the expanded population was CD56(+) NK cells; these effector cells showed significant up-regulation of the activating receptors NKG2D and DNAM-1. Functional tests demonstrated that expanded NK cells are fully functional with no difference whether tested before cryopreservation or after thawing.

Discussion: These data provide the basis for developing new NK-cell-based immunotherapeutic strategies for the treatment of patients with cancer.

Figure 1

Clinical grade NK-cell isolation and expansion. PBMC: peripheral blood mononuclear cells; NK: natural killer; GMP: good manufacturing practice.

Figure 2

Phenotypic analysis of NK cells obtained after performing the isolation procedure compared to the phenotype after expansion (one representative case). NK: natural killer.

Figure 3

(A) Changes in the phenotypic expression pattern on NK cells following expansion with cytokines plus autologous feeder. Results are expressed as mean fluorescence intensity ± standard deviation. (B) Levels of NKG2D and DNAM1 expression before and after expansion with cytokines alone or with cytokines plus autologous feeder (one representative case). *p≤0.05. NK: natural killer.

Figure 4

CD107a expression on cryopreserved expanded NK cells after stimulation with PMA and ionomycin (I) and in the presence of the K562 cell line. Each bar represents the mean percentage of triplicate measurements ± standard deviation. NK: natural killer; PMA: phorbol-12-myristate-13-acetate; E:T: effector:target.

Figure 5

Comparison between the lytic activity against the K562 cell line of fresh and cryopreserved expanded NK cells. Data are expressed as mean percentage of lysis ± standard deviation of four independent experiments. NK: natural killer; E:T: effector:target.