MASS PROLIFERATION CULTURE METHOD OF NK CELLS

Abstract

The present invention relates to a culture method of NK cell, the method comprising: 1) isolating a pellet of peripheral blood mononuclear cell (PBMC) containing immune cells from blood by using ficoll and centrifugation; 2) culturing the isolated PBMC in an anti-CD16 antibody-coated incubator in a medium containing RPMI culture medium, plasma, IL-2, IL-15, anti-CD56, anti-NKp46, and anti-NKp30; 3) subculturing the cultured PBMC in RPMI medium, albumin, IL-2, IL-15, anti-CD56, anti-NKp46, and anti-NKp30; and 4) treating the subcultured PBMC in a medium composition based on RPMI to which anti-NKp30, anti-NKp46 and anti-CD56 antibodies are added with a cytokine selected from a cytokine group consisting of IL-2, IL-15, and IL-2+IL-15 to activate NK cells. The present invention is an optimal combination that can effectively activate cells when NK cells are treated with IL-2 and IL-15 among various cytokines, and when treated with IL-2+IL-15, mass culture of NK cells is possible. Since apoptosis or killing ability of cancer cells can be promoted when this is used, the present invention can be used as an effective immune cell therapy for preventing or treating cancer.

Claims

1. A culture method of NK cell, the method comprising: 1) isolating a pellet of peripheral blood mononuclear cell (PBMC) containing immune cells from blood by using ficoll and centrifugation; 2) culturing the isolated PBMC in an anti-CD16 antibody-coated incubator in a medium containing RPMI culture medium, plasma, IL-2, IL-15, anti-CD56, anti-NKp46, and anti-NKp30; 3) subculturing the cultured PBMC in RPMI medium, albumin, IL-2, IL-15, anti-CD56, anti-NKp46, and anti-NKp30; and 4) treating the subcultured PBMC in a medium composition based on RPMI to which anti-NKp30, anti-NKp46 and anti-CD56 antibodies are added with a cytokine selected from a cytokine group consisting of IL-2, IL-15, and IL-2+IL-15 to activate NK cells.

2. The culture method of NK cell according to claim 1, wherein the cytokine of the Step 4) is treated with IL-2+IL-15.

3. The culture method of NK cell according to claim 2, wherein the cytokine of the Step 4) is treated with IL-2 20 ng/ml and IL-15 50 ng/ml.

4. The culture method of NK cell according to claim 1, wherein red blood cells are removed by treating an immune cell pellet with an RBC lysis buffer when isolating immune cells in the Step 1).

5. The culture method of NK cell according to claim 1, wherein the culturing in the Step 2) is carried out in a T25 flask coated with anti-CD16 antibody when the cell number is 310 7 or less and in a T75 flask coated with anti-CD16 antibody when the cell number is 310 7 or more by adding RPMI 8 ml, plasma 2 ml, IL-2 20 ng/ml, IL-15 50 ng/ml, anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, and anti-NKp30 5 ng/ml, suspending PBMC pellet in 10 ml RPMI and further adding the mixture, and culturing in an incubator at 37 C. and 5% CO.sub.2 for 3 to 4 days.

6. The culture method of NK cell according to claim 1, wherein the subculturing in the Step 3) is preferably carried out by adding the cell culture suspension of the Step 2) 20 ml, RPMI 72 ml, albumin 8 ml, IL-2 20 ng/ml, IL-15 50 ng/ml, anti-NKp30 5 ng/ml, anti-NKp46 5 ng/ml, and anti-CD56 5 ng/ml and culturing in an incubator at 37 C. and 5% CO.sub.2 for 3 to 5 days in primary subculture; and by adding primary subculture suspension 50 ml, RPMI 180 ml, albumin 20 ml, IL-2 20 ng/ml, IL-15 50 ng/ml, anti-NKp30 5 ng/ml, anti-NKp46 5 ng/ml, and anti-CD56 5 ng/ml and culturing in an incubator at 37 C. and 5% CO.sub.2 for 5 to 7 days in secondary subculture.

Description

DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A-1C show the results of flow cytometry according to the distribution (distribution of NK cells, NKT cells, and T cells) according to CD3 and CD56 antibodies for each of three types of immune cell PBMC samples isolated according to Example 1. The X-axis of the graph in FIG. 1 indicates CD3 (T lymphocyte marker) and the Y-axis indicates CD56 (NK lymphocyte marker).

[0025] FIGS. 2A-2b are graphs showing the results of measuring the amount of IFN- secretion according to the IL-2 and IL-15 cytokine treatment concentration in NK cells.

[0026] FIG. 3 is a graph showing the results of measuring the activity according to the amount of secretion of IFN- after setting the optimal concentrations of IL-2 and IL-15 cytokines according to FIGS. 2A-2B and treating the same at the set concentrations.

[0027] FIG. 4A-4E are graphs showing the results of measuring the distribution of immune cells according to antibody and IL-2 and IL-15 cytokine treatment under different conditions.

[0028] FIGS. 5A-5B are graphs showing the total cell count and cell viability of immune cells according to antibody and IL-2 and IL-15 cytokine treatment under different conditions.

[0029] FIG. 6 is a graph showing the number of cells measured after culturing NK cells for 14 days under set conditions.

[0030] FIG. 7 is a graph showing the results of FACS analysis test according to the NK cell mass culture composition under set conditions.

MODE OF THE INVENTION

[0031] Hereinafter, the present invention will be described in more detail through Examples. These Examples are only for explaining the present invention in more detail, and it will be obvious to one of ordinary skill in the art that the scope of the present invention is not limited by these Examples according to the gist of the present invention.

<Example 1> Isolation of Immune Cells from Blood

[0032] To isolate immune cells from blood, ficoll (GE Healthcare), PBS (WELGENE), two filter tubes (Greiner bio-one), four 50 ml tubes (VWR), an EDTA tube (BD biosciences), and a hematocytometer (MARIENFELD) were first prepared.

[0033] Blood of 50 to 100 ml, collected in a sterilized EDTA tube, was transported to a preparation room, and the blood was divided into 50 ml tubes. Ficoll of 15 ml each was put into two filter tubes and centrifuged at 2000 rpm for 1 minute, and the blood was divided into the prepared filter tubes containing the ficoll. The mixture was centrifuged at 2500 rpm for 25 minutes. Here, only the peripheral blood mononuclear cell (PBMC) layer was separated, collected in a new 50 ml tube, adjusted to a volume of 50 ml with PBS, and centrifuged at 2000 rpm for 10 minutes. When there were many RBCs (red blood cells) in a pellet, an RBC lysis buffer was used.

[0034] After centrifugation, the supernatant was removed, and the pellet was released with 10 ml of RPMI (culture medium) (Hyclone). Thereafter, an appropriate amount of suspension was taken and used for cell counting, and after adjusting to a volume of 50 ml with RPMI culture medium, the remaining suspension was centrifuged at 2000 rpm for 10 minutes.

[0035] At this time, the RBC lysis buffer was as described below.

[0036] The RBC lysis buffer of 1 ml was added per 110 8 to 210 8 cells and tapped for 1 minute. After adding 15 to 20 ml of RPMI (culture medium) thereto, centrifugation was performed at 1500 rpm (250 to 500G) for 7 minutes. When RBC removal was required further, the process described above was repeated 2 to 3 times.

<Example 2> Sample Peripheral Blood Mononuclear Cell (PBMC) Immune Cell Distribution

[0037] As described in Example 1 above, 40-60 cc of peripheral blood was divided into two filter tubes containing ficoll and then centrifuged at 2500 rpm for 25 minutes. Only the peripheral blood mononuclear cell (PBMC) layer was separated from the layered peripheral blood, collected in a new 50 ml tube, and washed with PBS. Cells obtained by discarding the supernatant were subject to a reaction with antibodies (CD56-PE, CD3-BV421) containing a fluorescent material and then analyzed by flow cytometry. Table 1 and FIGS. 1A-1C below show the results of immune cell distribution according to the antibodies.

TABLE-US-00001 TABLE 1 T NK NKT (CD3+CD56) (CD3CD56+) (CD3+CD56+) 1 Sample A 61.4 6.39 2.6 2 Sample B 73.3 5.63 6.72 3 Sample C 76.5 5.54 5.89

[0038] As shown in Table 1 and FIGS. 1A-1C above, it was confirmed that the overall distribution of the PBMC immune cells isolated from blood was NK cells 5% to 6%, NKT cells 2% to 7%, and T cells 60% to 80%.

<Example 3> NK Activity Measurement Test (IFN- Measurement) for Setting IL-2 and IL-15 Cytokine Concentrations

[0039] To confirm the effect and set the concentration of IL-2 and IL-15 cytokines as a preliminary experiment, monocytes obtained from peripheral blood according to Example 1 above were treated with respective concentrations of IL-2 and IL-15 cytokines in a flask coated with anti-CD16 antibody and in a medium containing RPMI 18 ml, plasma 2 ml, anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, and anti-NKp30 5 ng/ml and cultured for 7 days, and then all the cells were harvested. The collected cells were centrifuged at 2000 rpm for 5 minutes, and 1 ml of the supernatant was transferred to a new tube to measure cell activity. The cell activity was confirmed by measuring the amount of IFN- secretion by using an IFN- ELISA kit.

[0040] FIGS. 2A-2B show the amount of IFN- secretion according to the concentration of IL-2 and IL-15 treatment in NK cells. FIG. 3 shows the activity according to the amount of secretion of IFN- after the treatment at the optimal IL-2 and IL-15 concentrations set by the results shown in FIGS. 2A-2B.

[0041] Specifically, FIGS. 2A-2B show that the amount of secretion of IFN- gradually increased when the IL-2 treatment concentration was 5, 10, 15, 20, or 40 (ng/ml), and the highest secretion was reached at 20 ng/ml. The secretion did not increase any further at 40 ng/ml. On the other hand, when the IL-15 concentrations of 10, 30, 50, and 100 (ng/ml) were compared, the secretion of IFN- showed the highest secretion at 50 ng/ml and did not increase any further at 100 ng/ml. The results from the two experiments confirmed that 20 ng/ml of IL-2 and 50 ng/ml of IL-15 are the most effective concentration in terms of cell activity.

[0042] FIG. 3 shows that the amount of IFN- secretion in the cultured cells treated with IL-2+IL-15 was significantly higher than the amount of IFN- secretion in the cells treated with each of IL-2 and IL-15, confirming that the cell activity was increased.

<Example 4> Measurement of Immune Cell Distribution, Cell Viability, and Cell Count According to IL-2 and IL-15 Treatment

[0043] As a preliminary experiment for NK cell mass culture, to see the effect of each condition of antibody and IL-2 and IL-15 on NK cell ratio and cell viability or cell count, monocytes obtained from peripheral blood according to Example 1 above were treated under different conditions in a flask coated with anti-CD16 antibody and in a medium containing RPMI 18 ml and plasma 2 ml and then cultured for 7 days, and then all the cells were harvested to analyze the surface antigens of the NK cells. The treatment was performed for each of the group basically containing RPMI 18 ml and plasma 2 ml in a flask coated with anti-CD16 antibody to which only antibodies (anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, anti-NKp30 5 ng/ml) were added; the group basically containing RPMI 18 ml and plasma 2 ml in a flask coated with anti-CD16 antibody to which only IL-2 (20 ng/ml)+IL-15 (50 ng/ml) cytokines were added; the group basically containing RPMI 18 ml and plasma 2 ml in a flask coated with anti-CD16 antibody to which only antibodies (anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, anti-NKp30 5 ng/ml)+IL-2 (20 ng/ml) were added; the group basically containing RPMI 18 ml and plasma 2 ml in a flask coated with anti-CD16 antibody to which only antibodies (anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, anti-NKp30 5 ng/ml)+IL-15 (50 ng/ml) were added; and the group basically containing RPMI 18 ml and plasma 2 ml in a flask coated with anti-CD16 antibody to which antibodies (anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, anti-NKp30 5 ng/ml)+IL-2 (20 ng/ml)+IL-15 (50 ng/ml) were added, and the culture was performed for 7 days. Then, the culture was centrifuged at 2000 rpm for 10 minutes, and the cells obtained by removing the supernatant were subjected to a reaction with antibodies (CD56-PE, CD3-BV421) containing a fluorescent substance and analyzed by flow cytometry. The cell count was measured by using a hemocytometer after suspending the cells obtained by centrifugation in PBS and taking 10 l of the cell suspension and mixing the same with 10 l of Trypan Blue. Table 2 and FIGS. 4A-4E below show the results of the immune cell distribution according to the antibodies, and FIGS. 5A-5B show the total cell count and cell viability.

TABLE-US-00002 TABLE 2 T NK NKT (CD3+CD56) (CD3CD56+) (CD3+CD56+) Antibodies 67.0 11.2 7.10 IL-2 + IL-15 65.8 18.1 14.6 Antibodies + IL-2 60.1 19.7 16.6 Antibodies + IL-15 57.6 23.0 11.1 Antibodies + IL-2 + 38.2 40.4 20.1 IL-15

[0044] As shown in Table 2 and FIGS. 4A-4E, the ratio of NK cells was higher in the mixture treated with IL-2+IL-15 cytokine than in the sample treated with only the antibodies. However, it was confirmed that treating with the mixture prepared by combining antibodies+IL-2+IL-15 was effective in increasing NK and NKT cells. In addition, as shown in FIGS. 5A-5B, it was confirmed that the antibodies+IL-2+IL-15 mixture showed a proliferation effect 2 to 4 times higher than other control groups, and the viability was also maintained high.

<Example 5> NK Immune Cell Mass Culture Method at a Set Concentration

[0045] <5-1> Anti-CD16 Coating

[0046] Anti-CD16 was coated by mixing 10 ml of PBS and 5 ng/ml of anti-CD16 in a T75 flask, leaving the resulting mixture at room temperature for 6 hours, and then storing the same in a refrigerator overnight (24 h). The coated flask should be used within one week and was used after removing PBS and washing once with PBS before use. Since the flask is coated, care should be taken not to dry the bottom. Pipetting was carefully performed not to pipette directly to the bottom of the flask.

[0047] <5-2> Immune Cell Culture

[0048] First, in addition to the anti-CD16 coated T75 flask prepared in Example 5-1 above, RPMI medium, plasma, IL-2, IL-15, anti-CD56, anti-NKp46, and anti-NKp30 were prepared.

[0049] The cells were cultured in a T25 flask coated with anti-CD16 antibody when the number of cells in the cell suspension was 310 7 or less, and in a T75 flask coated with anti-CD16 antibody when the number was 310 7 or more. Specifically, RPMI 8 ml, plasma 2 ml, IL-2 20 ng/ml, IL-15 50 ng/ml, anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, and anti-NKp30 5 ng/ml were added to a coated T75 flask, and cells were obtained by the method of Example 1 by removing the supernatant. After pipetting with 10 ml of RPMI, the cells were added to the flask and cultured in an incubator at 37 C. and 5% CO.sub.2 for 3 to 4 days according to the conditions of the cells.

[0050] <5-3> Subculture

[0051] For subculture, a T175 or T225 flask, RPMI medium, albumin, IL-2, IL-15, anti-CD56, anti-NKp46, and anti-NKp30 were prepared.

[0052] <5-3-1> Primary Subculture (Subculture from T75 Flask to T175 Flask)

[0053] The immune cells cultured in the T75 flask of Example 5-2 above were subcultured into a T175 flask. Specifically, the T75 flask was scraped with a scraper to detach cells attached to the bottom, and the clumped cells were released by pipetting. The prepared cell suspension 20 ml was added to a new T175 flask with RPMI 72 ml, albumin 8 ml, IL-2 20 ng/ml, IL-15 50 ng/ml, anti-NKp30 5 ng/ml, anti-NKp46 5 ng/ml, and anti-CD56 5 ng/ml and mixed with the culture medium. Then, the cells were cultured in an incubator at 37 C. and 5% CO.sub.2 for 3 to 5 days according to the conditions of the cells.

[0054] <5-3-2> Secondary Subculture (Subculture from T175 Flask to Two T225 Flasks)

[0055] Subculture was performed from a T175 flask to two T225 flasks. Specifically, the T175 flask was scraped with a scraper to separate the cells attached to the bottom, and the clumped cells were released by pipetting. The prepared cell suspension of 100 ml was added to each of two new flasks, 50 ml each, with RPMI 180 ml, albumin 20 ml, IL-2 20 ng/ml, and IL-15 50 ng/ml, and mixed with the culture medium. Then, the cells were cultured in an incubator at 37 C. and 5% CO.sub.2 for 5 to 7 days according to the conditions of the cells. At this time, the media was added according to the cell conditions, and care was taken not to exceed the final volume of 400 ml for each T225 flask.

<Example 6> Cell Number Measurement Test after Culturing NK Cells for 14 Days at a Set Concentration

[0056] It was investigated whether the composition set for mass proliferation could allow for mass proliferation of NK cells and increase the distribution even after 14 days of culture. Specifically, 510 7 lymphocytes isolated by the method of Example 1 above were added to the culture medium of each sample, additionally treated with the set composition, which was RPMI+albumin+antibodies (anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, anti-NKp30 5 ng/ml)+IL-2 (20 ng/ml)+IL-15 (50 ng/ml), and then cultured for 14 days. In addition, the control groups were the group treated with only RPMI+albumin+IL-2 (20 ng/ml)+IL-15 (50 ng/ml) cytokines and the group treated with only RPMI+albumin+antibodies (anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, anti-NKp30 5 ng/ml). In addition, the cells were subcultured according to the number of cells and cultured for 14 days by adding a culture medium. For cell counting, the cultured cells were harvested on each day, 10 l of the suspended cells was taken and mixed with 10 l of Trypan Blue, and then measurement was performed with a hemocytometer. FIG. 6 shows the results. As shown in FIG. 6, when cultured for 14 days in the prepared culture medium, the cells were proliferated to more than 210 9 cells.

<Example 7> FACS Analysis Test According to the Composition of NK Cell Mass Culture at a Set Concentration

[0057] In the experiment (FACS) about the distribution of lymphocyte immune cells cultured according to Example 5-3-2, the cells were treated with RPMI+albumin+antibodies (anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, anti-NKp30 5 ng/ml)+IL-2 (20 ng/ml)+IL-15 (50 ng/ml) and cultured for 14 days, and the control groups were treated with only RPMI+albumin+IL-2 (20 ng/ml)+IL-15 (50 ng/ml) cytokines or with only RPMI+albumin+antibodies (anti-CD56 5 ng/ml, anti-NKp46 5 ng/ml, anti-NKp30 5 ng/ml), and each of the control groups was subcultured according to the number of cells for 14 days for comparison. The cells obtained by centrifugating at 2000 rpm for 10 minutes and removing the supernatant were subject to a reaction with antibodies (CD56-PE, CD3-BV421) containing a fluorescent material and analyzed by using flow cytometry. The results are shown in Table 3 and FIG. 7 below.

TABLE-US-00003 TABLE 3 T NK NKT (CD3+CD56) (CD3CD56+) (CD3+CD56+) Antibodies + IL-2 + 8.43 83.2 1.53 IL 15 IL-2 + IL15 35.1 42.4 21.1 Antibodies 52.5 33.2 13.5

[0058] As shown in Table 3 and FIG. 7 above, the results showed that the composition set for mass proliferation included 85% of NK and NKT cells and 8.5% of T cells even after 14 days of the mass culture, confirming that the NK cell ratio was significantly higher compared to the other control groups.

[0059] The present invention described above in detail with the preferred Examples of the present invention is not limited to the Examples above, and various modifications are possible by one of ordinary skill in the art within the scope of the technical principle of the present invention.