GMP-compliant, large-scale expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells in vitro and in vivo

Abstract

Ex vivo-expanded, allogeneic natural killer (NK) cells can be used for the treatment of various types of cancer. In allogeneic NK cell therapy, NK cells from healthy donors must be expanded in order to obtain a sufficient number of highly purified, activated NK cells. In the present study, we established a simplified and efficient method for the large-scale expansion and activation of NK cells from healthy donors under good manufacturing practice (GMP) conditions. After a single step of magnetic depletion of CD3(+) T cells, the depleted peripheral blood mononuclear cells (PBMCs) were stimulated and expanded with irradiated autologous PBMCs in the presence of OKT3 and IL-2 for 14 days, resulting in a highly pure population of CD3(-)CD16(+)CD56(+) NK cells which is desired for allogeneic purpose. Compared with freshly isolated NK cells, these expanded NK cells showed robust cytokine production and potent cytolytic activity against various cancer cell lines. Of note, expanded NK cells selectively killed cancer cells without demonstrating cytotoxicity against allogeneic non-tumor cells in coculture assays. The anti-tumor activity of expanded human NK cells was examined in SCID mice injected with human lymphoma cells. In this model, expanded NK cells efficiently controlled lymphoma progression. In conclusion, allogeneic NK cells were efficiently expanded in a GMP-compliant facility and demonstrated potent anti-tumor activity both in vitro and in vivo.

Figure 1. Characterization of large-scale GMP-expanded NK cells.

(A–B) T-cell depleted PBMCs were expanded under the GMP conditions described in the Materials and Methods. The percent of CD3⁻CD56⁺, CD56⁺CD16⁺, CD3⁺, CD14⁺ and CD19⁺ cells were analyzed by flow cytometric analyses (B, n = 8). Representative FACS dot plots are presented (A). (C) The fold expansion of NK cells was determined before (D0) and after (D14) NK cell expansion (n = 8). (D) The viability of expanded NK cells was evaluated by staining of propidium iodide. (E) Cytotoxicity of NK cells against various tumor cells was compared before (D0) and after (D14) NK cell expansion (n = 4). The effector∶target ratio was 10∶1. (F) NK cells were cocultured with K562 cells at 1∶1 ratio for 4 h, and staining for intracellular cytokines (IFN-γ and TNF-α) and CD107a was performed as described in the Materials and Methods. The data were compared before (D0) and after (D14) expansion. Mean and SD are presented. *p<0.05; **p<0.01; ***p<0.001.

Figure 2. Phenotypic comparisons of resting and expanded NK cells.

Surface expression of activating receptors (A), inhibitory receptors (B), activation markers (C) and chemokine receptors (D) was analyzed by flow cytometry before (D0) and after (D14) NK cell expansion (n = 10∼12). Individual or coexpression of KIRs (CD158a, CD158b or CD158e) was calculated by Boolean gates using FlowJo software (B). *p<0.05; **p<0.01; ***p<0.001.

Figure 3. Susceptibility of tumor cells to cytotoxicity of expanded NK cells.

(A) Cytotoxicity of expanded NK cells against various tumor cell lines was analyzed by ⁵¹Cr-release assay with the indicated effector∶target (E∶T) ratio in triplicate. Cytotoxicity against normal PBMCs was also analyzed. The assay was performed two times with expanded NK cells from different donors, and representative data was presented. Each graph represents mean±SD. (B) Expression of HLA-class I, ULBP-1, ULBP-2, MIC-A/B, CD112 and CD155 was analyzed by flow cytometry in various tumor cell lines and normal PBMCs (solid lines). Grey histograms represent isotype controls. (C) Expanded NK cells were pre-incubated with blocking antibodies for DNAM-1, NKG2D, NKp30 and/or NKp44, and cytotoxicity was analyzed against SW480 or SNU398 cells by ⁵¹Cr-release assay in triplicate. E∶T ratio was 10∶1. Percent inhibition of cytotoxicity was calculated as a percentage of the inhibition by the isotype control antibody. The assay was performed two times with expanded NK cells from different donors, and representative data are presented. Each bar graph represents mean+SD.

Figure 4. Cytotoxic activity of expanded NK cells against non-tumor cells.

Selective cytotoxicity of expanded NK cells against mixed targets of normal PBMCs and K562 cells was analyzed by flow cytometric cytotoxicity assay as described in the Materials and Methods. K562 tumor target cells were labeled with calcein-AM, and either allogeneic (A) or autologous (B) PBMC target cells were labeled with anti-HLA-class I. The expanded NK cells, the labeled K562 target cells, and the labeled PBMC target cells were cocultured at the ratio of 3∶1∶1 for 2 h. Dead cells were stained with 7-AAD, and the percent specific lysis was calculated. Cytotoxicity against PBMCs (left of each panel) and K562 cells (right of each panel) is presented separately. The assay was performed in duplicated. Each bar graph represents mean+SD.

Figure 5. In vivo distribution and anti-tumor efficacy of expanded NK cells in SCID mice.

(A) CFSE-labeled NK cells (2×10⁷ cells/mouse) were intravenously injected into SCID mice. Mice were sacrificed at 2, 24, 48, 72 and 168 h, and the percentage of CFSE⁺ cells in lungs, spleen, peripheral blood and liver was analyzed in lymphogating by flow cytometry (n = 4). Each graph represents mean±SEM. (B–C) SCID mice were injected intravenously in the tail vein with 1×10⁵ Raji cells and 1×10⁷ expanded NK cells in 400 µL of PBS on day 0 (n = 10/group). Three additional doses of expanded NK cells (1×10⁷cells/mouse) were administered within nine days. The monoclonal anti-CD20 antibody, rituximab (0.01 µg/mouse) was subcutaneously injected at the time of the first administration of expanded NK cells. Tumor-associated paralysis (B) and survival (C) were monitored. The efficacy test was confirmed by additional set of experiment using 10 mice per each group, and the representative set of the data is presented.