METHOD FOR PREPARING NATURAL KILLER CELLS USING IRRADIATED PBMCS, AND ANTI-CANCER CELL THERAPEUTIC AGENT COMPRISING THE NK CELLS

Abstract

Provided is a method for preparing natural killer cell with high efficiency using irradiated peripheral blood mononuclear cells, more particularly to a method for proliferating highly activated NK cells using a combination of irradiated peripheral blood mononuclear cells (PBMCs) and a CD16 antibody and an anti-cancer cell therapeutic composition containing the natural killer cells (NK cells) prepared thereby. Further provided is a method for large-scale proliferation of activated NK cells with high efficiency using a combination of irradiated peripheral blood mononuclear cells (PBMCs) and a CD16 antibody without the use of cancer cells or genetically modified feeder cells having safety issues as feeder cells. The highly purified and highly cytotoxic NK cells proliferated in large quantities can be used as an active ingredient of a cancer immunotherapeutic composition.

Claims

1. A method for preparing highly purified activated natural killer cells (NK cells) using feeder cells, wherein irradiated peripheral blood mononuclear cells (PBMCs) are used as the feeder cells and the NK cells are treated with a CD16 antibody.

2. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, which comprises: a) isolating peripheral blood mononuclear cells (PBMCs) from human peripheral blood; b) isolating natural killer cells (NK cells) from the isolated peripheral blood mononuclear cells; c) preparing feeder cells by irradiating the peripheral blood mononuclear cells (PBMCs) remaining after isolating the natural killer cells; and d) culturing the isolated natural killer cells (NK cells) and the prepared feeder cells in a CD16 antibody-immobilized incubator.

3. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 2, wherein, in b), the natural killer cells (NK cells) are isolated from the isolated peripheral blood mononuclear cells using a magnetic microbead-attached antibody and a column.

4. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 2, wherein, in c), the feeder cells are prepared by mixing the peripheral blood mononuclear cells (PBMCs) remaining after isolating the NK cells well in physiological saline or a medium and irradiating at 23-27 Gy.

5. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 2, wherein, in d), the isolated NK cells are treated with NKG2D and 2B4 antibodies.

6. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, wherein the irradiated peripheral blood mononuclear cells (PBMCs) inhibits the activation of T cells and increases the expression of NKG2D ligands and CD48.

7. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, wherein the proliferation of the NK cells is promoted by a combination of the irradiated peripheral blood mononuclear cells (PBMCs) and the CD16 antibody.

8. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, wherein the proliferation of the NK cells is strongly induced by a synergistic combination of activating receptors CD16, NKG2D and 2B4

9. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, wherein the expression of activating receptors of the NK cells is increased by a combination of the irradiated peripheral blood mononuclear cells (PBMCs) and the CD16 antibody.

10. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, wherein CD107a is highly expressed in the NK cells proliferated by a combination of the irradiated peripheral blood mononuclear cells (PBMCs) and the CD16 antibody.

11. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, wherein the NK cells proliferated by a combination of the irradiated peripheral blood mononuclear cells (PBMCs) and the CD16 antibody strongly increases the secretion of IFN-? upon stimulation by target cancer cells.

12. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, wherein the NK cells proliferated by a combination of the irradiated peripheral blood mononuclear cells (PBMCs) and the CD16 antibody show strongly increased antitumor cytotoxicity against target cancer cells.

13. The method for preparing highly purified activated natural killer cells (NK cells) according to claim 1, wherein the NK cells proliferated by a combination of the irradiated peripheral blood mononuclear cells (PBMCs) and the CD16 antibody show strong antitumor effect in a cancer-induced mouse model.

14. An anti-cancer cell therapeutic composition comprising highly purified activated natural killer cells (NK cells) prepared by the method according to claim 1 as an active ingredient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

[0048] FIG. 1A shows a result of irradiating PBMCs with various radiation doses (5, 10, 15, 20 and 25 Gy), culturing them with NK cells and measuring the proportion of NK cells and T cells by flow cytometry.

[0049] FIG. 1B shows a result of irradiating PBMCs at a radiation dose of 25 Gy, measuring the expression of NKG2D ligands with time by flow cytometry and representing the relative expression ratio as compared to before the radiation.

[0050] FIG. 1C shows a result of irradiating PBMCs at a radiation dose of 25 Gy, measuring the expression of CD48 with time by flow cytometry and representing the relative expression ratio as compared to before the radiation.

[0051] FIG. 2A shows a result of measuring cell proliferation with the Cell Counting Kit-8 (CCK-8) using blocking antibodies specific to each receptor in order to confirm whether the proliferation of NK cells is due to the synergistic combinations of activating receptors CD16, NKG2D and 2B4.

[0052] FIG. 2B shows a result of investigating the proliferation of NK cells for 21 days using irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD16) either alone or in combination.

[0053] FIG. 3 shows a result of proliferating NK cells using irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD16) either alone or in combination and then measuring the expression level of activating receptors by flow cytometry.

[0054] FIG. 4A shows a result of proliferating NK cells using irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD16) either alone or in combination, culturing them with cancer cells (K562) and then measuring the expression level of CD107a by flow cytometry.

[0055] FIG. 4B shows a result of proliferating NK cells using irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD116) either alone or in combination, culturing them with cancer cells (K562) and then measuring the expression level of CD1007a by flow cytometry.

[0056] FIG. 5 shows a result of proliferating NK cells using irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD16) either alone or in combination, culturing them with cancer cells (K562) and then measuring IFN-? secretion by the enzyme-linked immunospot (ELISpot) assay.

[0057] FIG. 6A shows a result of proliferating NK cells using irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD16) either alone or in combination and then measuring the cytotoxicity against cancer cells (K562) by flow cytometry.

[0058] FIG. 6B shows a result of measuring the expression of NKG2D ligands in various cancer cells by flow cytometry.

[0059] FIG. 6C shows a result of proliferating NK cells using a combination of irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD16) and then measuring the cytotoxicity against various cancer cells by flow cytometry.

[0060] FIG. 7A shows the antitumor effect of NK cells proliferated using a combination of irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD16) in colon and lung cancer NOD/SCID mouse models.

[0061] FIG. 7B shows the expression level of NKG2D ligands in irradiated colon and lung cancer cells and the cytotoxicity of proliferated NK cells (using a combination of irradiated PBMCs and an anti-CD16 monoclonal antibody (?CD16)).

MODE FOR INVENTION

[0062] Practical and presently preferred embodiments of the present invention are illustrated as shown in the following examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1. Culturing of Cancer Cell Lines

[0063] K562 (CCL-243), SW480 (CCL-288), A549 (CCL-185) and MCF-7 (HTB-22) cells were cultured in RPMI 1640 (K562, SW480, A549) or DMEM (MCF-7) supplemented with 100 U/mL penicillin, 100 ?g/mL streptomycin and 10% fetal bovine serum (FBS) in a 5% CO.sub.2 incubator maintained at 37? C.

Example 2. Isolation and Culturing of NK Cells

[0064] 1) Separation of Blood

[0065] 10-50 mL of human peripheral blood was centrifuged (2000 rpm, 5 minutes). From the separated blood, the supernatant plasma and the red blood cells which settled down were discarded and the white blood cells in the middle layer were recovered. The recovered white blood cells were mixed well by adding physiological saline (normal saline) and loaded the density gradient solution Histopaque-1077. Then, peripheral blood mononuclear cells (PBMCs) were obtained by centrifuging at 400?g for 30 minutes at room temperature.

[0066] 2) Isolation of NK Cells

[0067] Highly purified natural killer cells were obtained by incubating the isolated peripheral blood mononuclear cells with magnetic microbead-attached antibodies such as a CD56 antibody (for positive selection) or CD3, CD14 and CD19 antibodies (for negative selection) in a column.

[0068] 3) Preparation of Feeder Cells

[0069] After the isolation of the NK cells, the remaining peripheral blood mononuclear cells (PBMCs) were mixed well in physiological saline (or a medium) and irradiated at a radiation dose of 25 Gy.

[0070] 4) Preparation of Antibody-Immobilized Incubator

[0071] An anti-CD16 antibody prepared with a concentration of 1 ?g/mL or higher in physiological saline was added to an incubator and the solution was allowed to spread uniformly on the bottom. 4-24 hours later, the antibody solution was removed and the incubator was washed 3 times with physiological saline to obtain an antibody-immobilized incubator.

[0072] 5) Culturing of NK Cells

[0073] The natural killer cells (NK cells) and feeder cell (NK cells: feeder cells=1:1-100) isolated from the peripheral blood mononuclear cells were mixed well in a medium and added to the antibody-immobilized incubator. After adding 5-10% human serum and 500-1000 U/mL interleukin-2 (Proleukin, Chiron), the cells were cultured for 3-7 days at 37? C. in the presence of 5% CO.sub.2. Then, the cells were transferred to an incubator with no antibody immobilized and a medium supplemented with 5-10% human serum and 500-1000 U/mL interleukin-2 (hereinafter referred to as a nutrient medium) was added. The cells were cultured for 21 days while adding the nutrient medium every 2-3 days depending on the degree of proliferation of the natural killer cells. On days 7, 14 and 21, the cells were recovered from the incubator in order to investigate the proliferation of the natural killer cells and identify surface antigens.

Example 3. Analysis of Surface Antigens

[0074] Surface antigens on the cells were analyzed using monoclonal antibodies for flow cytometry. Fluorescence-labeled monoclonal antibodies such as anti-CD3-PE, CD48-FITC, CD56-PE-Cy5, CD16-PE, CD314 (NKG2D)-PE, HLA-ABC-FITC, CD337 (NKp30)-PE, CD336 (NKp44)-PE, CD335 (NKp46)-PE, CD226 (DNAM-1)-FITC, CD244 (2B4)-FITC, MICA-PE, MICB-PE, ULBP-1-PE, ULBP-2-PE, ULBP-3-PE, etc. were used and analysis was conducted with respect to the isotype control.

Example 4. Confirmation of NK Cell Proliferation by Activating Receptors of NK Cells

[0075] The isolated NK cells were incubated with m1gG, NKG2D, CD244 (2B4) and NKG2D+CD244 (2B4) antibodies for 30 minutes in an incubator maintained at 37? C. and 5% CO.sub.2 and then washed 3 times with physiological saline. The antibody-bound NK cells were seeded onto a 96-well plate or a CD16 antibody-immobilized 96-well plate to a concentration of 1?10.sup.5 cells/mL. Then, the NK cells were cultured after adding feeder cells. After culturing for 5 days and adding 10 ?L of the CCK-8 (Cell Counting Kit-8) reagent to each well, the cells were incubated for 4 hours in an incubator maintained at 37? C. and 5% CO.sub.2. 4 hours later, absorbance was measured at 450 nm using an ELISA reader.

Example 5. Confirmation of NK Cell Function

[0076] 1) Analysis of CD107a

[0077] NK cells were cocultured with K562 (human chronic myelogenous leukemia cell line) cells at a ratio of 1:1 in a medium supplemented with anti-CD107a-PE, BD GolgiStop? and BD GolgiPlug? for 4-6 hours at 37? C. in the presence of 5% CO.sub.2. Then, the cells were centrifugally washed 3 times with physiological saline and then incubated with anti-CD56-PC5 for 20-30 minutes. Then, the expression level of CD1007a was measured by flow cytometry.

[0078] 2) Analysis of Interferon Gamma (IFN-?) by Enzyme-Linked Immunospot (ELISpot) Assay

[0079] NK cells and target cancer cells (1:10) were added to an ELISpot plate coated with a capture antibody and containing 200 ?L of a nutrient medium and then incubated for 4 hours in an incubator maintained at 37? C. and 5% CO.sub.2. After washing with physiological saline, a detection antibody was added at 100 ?L per well and the plate was incubated for 2 hours at room temperature. After washing with physiological saline, a color developing reagent was added to each well and the plate was incubated in the dark. After the incubation, the color developing reaction was completed using distilled water and the plate was dried well. Finally, interferon gamma (IFN-?) was quantified using the ELISpot reader system.

[0080] 3) NK Cell-Mediated Cytotoxicity Assay

[0081] In the present invention, K562, A549, SW480 and MCF-7 cells were used as the target cancer cells of NK cells. After adding 5 ?M 5-carboxyfluorescein diacetate succinmidyl ester (CFSE), the target cancer cells were incubated at 37? C. for 10 minutes in the presence of 5% CO.sub.2. Then, the cells were centrifugally washed 2-3 times using a medium supplemented with 10% human serum. NK cells (effector cells) were cocultured with the CFSE-labeled target cancer cells at ratios of 10:1, 5:1, 2.5:1 and 1:1 in a reactor tube or a 96-well plate for 4-6 hours at 37? C. in the presence of 5% CO.sub.2. After the culturing was completed, the tube was immediately put in ice water and 50 ?g/mL propidium iodide (PI) was added. The cytotoxicity of the natural killer cells (NK cells) was analyzed by flow cytometry within 1 hour.

Example 6. Animal Experiment of NK Cells

[0082] 5-to-6-week-old nonobese diabetic/severe combined immunodeficiency (NOD/SCID) NOD.CB17-Prkdcscid/ARC mice were used for animal experiment of NK cells. SW480 human colon cancer cells (2-5?10.sup.6 cells) and A549 human lung cancer cells (2-5?10.sup.6 cells) were subcutaneously inoculated into the right thighs of the mice. When the tumor grew to a size of 50-100 mm.sup.3, irradiation was applied at 8 Gy to the right thigh using a linear accelerator (Infinity. Elekta). After the irradiation, NK cells (1-2?10.sup.7 cells) were injected into the tail veins of the mice. The tumor size (volume=depth?width.sup.2?0.5) was measured twice a week and the irradiation and the NK cell injection were performed 3 times at 1-week intervals. 5-FU (100 mg/kg, SW480 positive control group) and docetaxel (10 mg/kg, A549 positive control group) were administered 3 days before every NK cell injection.

[0083] Experimental Results

[0084] Result 1. Irradiation Inhibits T Cell Activity and Increases Expression of NKG2D Ligands and CD48 in Peripheral Blood Mononuclear Cells (PBMC)

[0085] To determine the optimal dose of radiation for T-cell inactivation. PBMCs were exposed to various radiation doses (5, 10, 15, 20, 25 Gy). Then, the irradiated PBMCs were cocultured with resting NK cells (NK cells isolated from peripheral blood) for 21 days. The proportion of T cells was assessed by flow cytometry (FIG. 1A). T cells were clearly detectable during NK cell activation and proliferation after radiation doses of 5, 10, 15 and 20 Gy. However, the radiation dose of 25 Gy induced effective inactivation of T cells (T cells were hardly observed). Specifically, when NK cells were cocultured feeder cells irradiated at a dose of 25 Gy, the proportion of NK cells (green) was higher than 99% (99.84%) and the proportion of T cells (red) was less than 1% (0.12%). Therefore, 25 Gy was decided as the radiation dose for effectively inactivating T cells. To test whether irradiation induces the expression of NKG2D ligands and CD48 (2B4 ligand) in peripheral blood mononuclear cells, peripheral blood mononuclear cells isolated from donors were harvested 0, 24, 48 or 72 hours after irradiation at 25 Gy. The expression of NKG2D ligands (FIG. 1B) and CD48 (FIG. 1C) was analyzed by flow cytometry and the result was represented by mean fluorescence intensities (MFIs). Relative expression ratios were calculated by dividing the MFI value of the irradiated peripheral blood mononuclear cells by the MFI value of the fresh peripheral blood mononuclear cells. The irradiated peripheral blood mononuclear cells expressed larger amounts of MICA, ULBP3 and CD48 compared to fresh peripheral blood mononuclear cells after 2 days, whereas MICB, ULBP1 and ULBP2 expression increased 3 days after the irradiation. In addition, although the PBMCs highly expressed CD48 (2B4 ligand), this expression was further increased 2 days after the irradiation. These results indicate that the radiation dose of 25 Gy increases the expression of NKG2D ligands and CD48 in peripheral blood mononuclear cells.

[0086] FIG. 1A shows the result of irradiating PBMCs with various doses (5, 10, 15, 20 and 25 Gy), coculturing them with NK cell for 21 days and then measuring the proportions of NK cells and T cells by flow cytometry. In FIGS. 1B and 1C, the dotted lines indicate the MFI value of the peripheral blood mononuclear cells before the irradiation. Relative expression ratios were calculated by dividing the MFI value of the irradiated peripheral blood mononuclear cells by the MFI value of the fresh peripheral blood mononuclear cells. Statistical significance: *P<0.05, **P<0.005, ***P<0.0005.

[0087] Result 2. A Synergistic Combination of Activating Receptors CD16, NKG2D and 2B4 Strongly Induces Proliferation of NK Cells

[0088] To examine the effect of a combination of irradiated peripheral blood mononuclear cells (IrAP; cells in which NKG2D and 2134 are expressed) and an anti-CD16 monoclonal antibody (?CD16) on the proliferation of NK cells, resting NK cells (NK cells isolated from peripheral blood) from five donors were isolated and irradiated. ?CD16 was coated onto a plate to a concentration of 1 ?g/mL or higher in advance and resting NK cells and IrAPs were cultured under Good Manufacturing Practices (GMP) conditions. First, it was investigated whether the NK cell proliferation was due to the synergistic combinations of activating receptors CD16, NKG2D and 2B4 by the Cell Counting Kit-8 (CCK-8) assay using blocking antibodies specific for each receptor. Although IrAP strongly induced the proliferation of NK cells, the proliferation of NK cells was further enhanced by a combination of IrAP and ?CD16. However, the proliferation of NK cells was relatively low when ?CD16 was used alone as compared to IrAP or IrAP+?CD16 (FIG. 2A). It was confirmed that, for the NK cells treated with a combination of IrAP and ?CD16, treatment with NKG2D- or 2B4-blocking antibody resulted in significantly decreased proliferation of NK cells. In particular, the proliferation of NK cells was more strongly inhibited by the treatment with the NKG2D- and 2B4-blocking antibodies at the same time. These results indicate that the proliferation of NK cells is induced by coactivation of receptors NKG2D and 2B4, and this effect was more strongly induced by synergistic combinations of the receptors CD16, NKG2D and 2B4. In particular, it was confirmed that this effect is more strongly induced by the synergistic combinations of the activating receptors CD16, NKG2D and 2B4. As shown in FIG. 2B, IL-2 alone failed to significantly induce the proliferation of NK cells (42.8?3.8 fold), whereas the NK cells stimulated with IrAP or ?CD16 were significantly proliferated as compared to IL-2 alone (IrAP; 794?115.6 fold, ?CD16; 259.2?44.4 fold). In particular, the NK cells stimulated with a combination of IrAP and ?CD16 were remarkably proliferated (5421.6?505.4 fold). This interaction points to a synergistic effect of IrAP and ?CD16 in the proliferation of NK cells. Thus, it was demonstrated that a combination of IrAP and ?CD16 synergistically enhances the proliferation of NK cells. In FIG. 2, statistical significance: ###P<0.0005 (#; NK alone versus other groups). ***P<0.0005 (*; NK+IrAP versus NK+?CD16 or NK+?CD16+IrAP).

[0089] Result 3. A Combination of Irradiated Peripheral Blood Mononuclear Cells (IrAP) with an anti-CD16 Monoclonal Antibody (?CD16) Increases the Expression of NK Cell-Activating Receptors

[0090] The phenotypic differences between NK cells isolated from peripheral blood (resting NK cell) and proliferated NK cells were evaluated. These cells were analyzed by flow cytometry and then the expression levels of CD3, CD56, CD16, NKG2D (CD314), NKp30 (CD337), NKp44 (CD336), NKp46 (CD335), 2184 (CD244) and DNAM-1 (CD226) were compared. As shown in FIG. 3, the NK cells proliferated by a combination of IrAP and ?CD16 showed significantly increased expression of activating receptors (NKG2D, DNAM-1, 2B4, NKp30, NKp44 and NKp46) as compared to the resting NK cells. Nonetheless, CD3, CD56 and CD16 expression levels were not significantly changed. In addition, this proliferation method produced significant differences in CD3, CD56, CD116, DNAM-1, 2B4, NKp30, NKp44 and NKp46 as compared to the NK cells expanded by either IrAP or ?CD16 alone. The NK cells proliferated by either IrAP or ?CD16 showed significant differences in NKG2D, DNAM-L, 2B4/NKp46 (NK cells proliferated by ?CD16) and NKp44 as compared to the resting NK cells. In contrast, CD56 and CD16 expression levels significantly decreased and NKp30 showed no significant change. Furthermore, there were significant differences in the expression levels of DNAM-1, 284, NKp44 and NKp46 between the NK cells proliferated by IrAP and ?CD16. In addition, the NK cells proliferated by a combination of IrAP and ?CD16 had negligible T-cell (CD3) contamination as compared to the NK cells proliferated by either IrAP or ?CD16 alone. T cells were hardly detectable during the proliferation (<1%). Thus, these results indicate that the combination of IrAP and ?CD16 may further increase the expression of the NK cell-activating receptors in the proliferation of NK cells. In FIG. 3, statistical significance: #P<0.05, ##P<0.005, ###P<0.0005 (#; NK alone versus other groups). *P<0.05, **P<0.005, ***P<0.0005 (*; NK+IrAP versus NK+?CD16 or NK+?CD16+IrAP.

[0091] Result 4. CD107a is Highly Expressed in NK Cells Proliferated by a Combination of IrAP and ?CD16

[0092] It is known that CD107a expression correlates closely with the activity of NK cells [24]. It was determined whether the degranulation marker CD107a was expressed on the surface of the NK cells proliferated under various conditions. The proliferated NK cells were incubated with K562 cells as target cancer cells. After 4 hours of incubation in the presence of monensin and an anti-CD107a monoclonal antibody, NK cells were stained by adding anti-CD3 and anti-CD56 monoclonal antibodies. As shown in FIG. 4, the resting NK cells expressed very little CD107a on the cell surface upon contact with the K562 cells, but CD107a expression on the surface of the NK cells (proliferated under various culture conditions) increased more than 2.7-fold as compared to the resting NK cells. In particular, the CD107a expression on the surface of NK cells proliferated by a combination of IrAP and ?CD16 was 6.1-fold as compared to the resting NK cells. Thus, these results indicate that the NK cells proliferated by a combination of IrAP and ?CD16 may further increase the expression of CD107a caused by stimulation with target cancer cells. In FIG. 4, statistical significance: ##P<0.005, ###P<0.0005 (#; NK alone versus other groups). **P<0.005, (*; NK+IrAP versus NK+?CD16 or NK+?CD16+IrAP).

[0093] Result 5. NK Cells Proliferated by a Combination of IrAP and ?CD16 Strongly Increase IFN-? Secretion after Stimulation with Target Cancer Cells

[0094] The IFN-? secretion of NK cells after stimulation with target cancer cells was evaluated. The IFN-? ELISpot assay was performed on resting NK cells (NK cells isolated from peripheral blood) and proliferated NK cells using K562 cells as target cancer cells. The resting NK cells secreted relatively low amounts of IFN-? after stimulation with K562 cells, but the NK cells proliferated under various culture conditions strongly increased IFN-? secretion. Specifically, the NK cells proliferated by a combination of IrAP and ?CD16 secreted larger amounts of IFN-? than did the NK cells proliferated by either IrAP or ?CD16. These results may be related to the CD107a expression. Thus, these findings indicate that the NK cells proliferated by a combination of IrAP and ?CD16 may further increase IFN-? secretion after stimulation with target cancer cells.

[0095] In FIG. 5, statistical significance: ##P<0.005, ###P<0.0005 (#; NK alone versus other groups). *P<0.05, **P<0.005, (*; NK+IrAP versus NK+?CD16 or NK+?CD16+IrAP).

[0096] Result 6. NK Cells Proliferated by a Combination of IrAP and ?CD16 Show Strongly Enhanced Antitumor Cytotoxicity Against Target Cancer Cells

[0097] The antitumor cytotoxicity of NK cells proliferated using an MHC class I-negative cell line (K562) and MHC class I-positive cell lines (MCF-7, A549, and SW480) was evaluated. As shown in FIG. 6A, the antitumor cytotoxicity against target cancer cells was significantly elevated in the proliferated NK cells compared to resting NK cells (NK cells isolated from peripheral blood) and NK-92 cells. In particular, the NK cells proliferated by a combination of IrAP and ?CD16 showed higher antitumor cytotoxicity than did the NK cells proliferated by either the IrAP or ?CD16. These results may be related to CD107a expression and IFN-? secretion. As shown in FIG. 6B, the most NK-sensitive target cancer cells, K562 cells, expressed NKG2D ligands but did not express MHC class I. The A549 cells weakly expressed NKG2D ligands but MHC class I was strongly expressed. They were weakly sensitive to the NK cells proliferated by a combination of IrAP and ?CD16. Although the MCF-7 and SW480 cells expressed MHC class I, these cells strongly expressed NKG2D ligands as compared to other cancer cells. They were moderately sensitive to the NK cells proliferated by a combination of IrAP and ?CD16. The NK-sensitive target cancer cells (K562, MCF-7 and SW480) tended to highly express NKG2D ligands or weakly express MHC class I as compared to the NK-resistant target cells (A549). To evaluate the effect of NKG2D ligands on the antitumor cytotoxicity of NK cells, the NK cells proliferated by a combination of IrAP and ?CD16 were cocultured with target cancer cells in the presence of a NKG2D-blocking antibody (used to inhibit the biding between the NKG2D receptors of NK cells and the ligands of the target cancer cells). Blocking of the receptor NKG2D resulted in a substantial reduction in antitumor cytotoxicity against all target cancer cells except for the A549 cells, which show low expression of NKG2D ligands (FIG. 6C). These results indicate that the NK cells proliferated by a combination of IrAP and ?CD16 exert increased antitumor cytotoxicity against target cancer cells and NKG2D is one of the important factors in the activation of NK cells. In FIG. 6, statistical significance: #P<0.05, ##P<0.005, ###P<0.0005 (#; NK alone versus other groups). *P<0.05, **P<0.005, ***P<0.0005 (*; NK+IrAP versus NK+?CD16 or NK+?CD16+IrAP, target tumor cell versus NKG2D blocking). @P<0.05, @@P<0.005 (@; NK+?CD16 versus NK+IrAP or NK+?CD16+IrAP).

[0098] Result 7. NK Cell Proliferated by a Combination of IrAP and ?CD16 Strongly Induce Antitumor Effect in Colon and Lung Cancer NOD/SCID Mouse Models

[0099] The antitumor effect of NK cells proliferated by a combination of IrAP and ?CD16 was evaluated using colon and lung cancer NOD/SCID mouse models. SW480 (human colon cancer) cells and A549 (human lung cancer) cells were subcutaneously inoculated into the right thighs of NOD-SCID mice. Irradiation was applied at a radiation dose of 8 Gy to the tumor in the right thigh of the mice. Then, the proliferated NK cells proliferated by a combination of IrAP and ?CD16 were injected into the tail vein. 5-FU and docetaxel were injected 3 days before every NK injection. The NK cells proliferated by a combination of IrAP and ?CD16 significantly inhibited tumor growth in both colon cancer (SW480) and lung cancer (A549) NOD/SCID mouse models. In particular, the antitumor effect of the NK cells was further enhanced by the combined treatment with irradiation (FIG. 7A). The irradiation increased the expression of NKG2D ligands in the SW480 and A549 cells and further enhanced the cytotoxicity of the NK cell against target cancer cells (FIG. 7B). These results demonstrate the in vivo antitumor effect of the NK cells proliferated by a combination of IrAP and ?CD16 in colon cancer (SW480) and lung cancer (A549) NOD/SCID mouse models. In particular, the combined treatment with irradiation could further enhance the in vivo antitumor activity of the NK cells by increasing the expression of NKG2D ligands in cancer cells. In FIG. 7, statistical significance: *P<0.05, **P<0.005, ***P<0.0005 (*; con versus other groups, 0 h 0 Gy versus 48 h 8 Gy). ###P<0.0005 (#; NK versus other groups). P<0.0005 (; IR versus NK+IR or docetaxel).

[0100] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

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