KR101035556B1 - Method of preparing natural killer cells, natural killer cells prepared by the method, and composition for treatment of solid cancer containing same - Google Patents

Abstract

The present invention is a method for producing natural killer cells using Interleukin-15 (IL-15), natural killer cells prepared by such a method, and treating solid tumors comprising the natural killer cells as an active ingredient. It relates to a composition for.

According to the method of the present invention, the natural killer cells having excellent solid tumor killing ability can be obtained by activating natural killer cells that are difficult to amplify in vitro ( ex vivo ), and the composition comprising the natural killer cells obtained by such a method as an active ingredient. May be useful for the treatment of solid tumors, including neuroblastoma.

Interleukin-15, natural killer cells, solid tumors

Description

METHOD OF PREPARING NATURAL KILLER CELLS, NATURAL KILLER CELLS PREPARED BY THE METHOD, AND COMPOSITION FOR TREATMENT OF SOLID CANCER CONTAINING SAME}

The present invention relates to a method for producing natural killer cells, and more specifically, to a method for producing natural killer cells using Interleukin-15 (IL-15), natural killer prepared by such a method. Cells, and relates to a composition for treating solid tumors comprising the natural killer cells as an active ingredient.

Various treatments such as surgery, radiation therapy, chemotherapy, etc. have been developed for the treatment of tumors, but frequent recurrences become serious problems depending on the tumor. This raises the possibility of cell therapy using the immune function of patients.

Immune response to tumor removal occurs through the complex interaction of immune cells with different functions. Immune cells that directly remove tumor cells include natural killer cells (NK cells) and cytotoxic T lymphocytes (CTL), and antigens that present antigens to these effect cells. Presenting cells include dendritic cells (DC) or B cells, and other T cytokines (Helper T cells, Th cells) and regulatory T cells (Regulatory T cells, Treg cells) ) Will contribute together. Of these, natural killer cells are considered to be the most effective and efficient immune cells among immune cell therapies.

Natural killer cell therapies that have been developed or tried to date have been confirmed by administration after allogeneic bone marrow transplantation to blood tumors such as leukemia (Blood cells, Molecules & Disease, 33: 261-266 (2004)). However, none of the clinical studies of solid tumors except blood tumors have demonstrated such therapeutic effects.

Specifically, natural killer cells have been reported to interfere with tumor engraftment prior to tumor development (Cancer immunology, immunotherapy, 56 (11): 1733-1742 (2007)). Some experiments have inhibited the growth of breast cancer cells by the administration of cells, but it is unclear whether the effect is caused by natural killer cells (Breast cancer research and treatment, 104 (3): 267-275 (2007)).

Natural killer cells make up about 10% of the blood cells, and because they do not grow well in vitro , there is a limit to using them in effective amounts.

Therefore, the present inventors continued to find a method for culturing a large amount of functionally excellent natural killer cells, and as a result, in vitro ( ex In vivo ) IL-15 was used to culture natural killer cells and when administered to a solid tumor-derived animal model, it was found that tumor cell killing ability was excellent and the present invention was completed.

An object of the present invention is to provide a method for preparing natural killer cells using Interleukin-15 (IL-15).

Another object of the present invention is to provide natural killer cells prepared using the above method and capable of inducing solid tumor specific immune responses.

Still another object of the present invention is to provide a composition for treating solid tumors comprising the natural killer cells as an active ingredient.

According to the above object, the present invention comprises the steps of: 1) separating the spleen cells of the mammal; 2) culturing for 3 to 14 days by adding IL-15 to the isolated splenocytes; And 3) isolating CD5 ^- cells from splenocytes.

In accordance with the above object, the present invention also provides a method for preparing a mononuclear cell, comprising the steps of: 1) separating monocytes from peripheral blood of a mammal, including human; 2) separating CD56 ⁺ monocytes from isolated peripheral blood monocytes; And 3) adding IL-15 to the isolated CD56 ⁺ monocytes, and culturing for 2 to 30 days to provide natural killer cells.

According to this other object, the present invention provides a natural killer cell prepared by the above method and capable of inducing a solid tumor specific immune response.

In accordance with another object, the present invention provides a composition for treating solid tumors, comprising natural killer cells as an active ingredient and comprising one or more pharmaceutically acceptable carriers or adjuvants.

According to the method of the present invention, the natural killer cells having excellent solid tumor killing ability can be obtained by activating natural killer cells that are difficult to amplify in vitro ( ex vivo ), and the composition comprising the natural killer cells obtained by such a method as an active ingredient. May be useful for the treatment of solid tumors, including neuroblastoma.

The terms, techniques, and the like described in the present invention are used as meanings generally used in the technical field to which the present invention belongs, unless otherwise specified. In addition, all the documents mentioned in this specification are included in this specification as the documents for demonstrating this invention.

In the present invention, "solid tumor (cancer)" refers to all tumors (cancer) composed of lumps except blood tumors (cancer), and typical examples thereof include melanoma, neuroblastoma, and the like.

The expression "activation of natural killer cells" means that the expression of NKG2D and the like on the surface of natural killer cells is increased, and the ability to kill tumor cells is significantly improved.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing natural killer cells (Natural Killer cells, NK cells) using Interleukin-15 (IL-15) and natural killer cells prepared by the method.

Natural killer cells of the present invention

1) isolating splenocytes of a mammal;

2) culturing for 3 to 14 days by adding IL-15 to the isolated splenocytes; And

3) Separating CD5 ^- monocytes from splenocytes

It can manufacture by the method containing.

In more detail, the splenocyte separation in step 1) can be performed by a conventional method. For example, splenocytes of a mouse can be obtained by aseptically removing the spleen from the mouse, washing with Hanks' balanced salt solution (HBSS), and cell down to remove supernatants and red blood cells. Splenocytes are most preferably used immediately after culturing spontaneous killer cells, but may be used after cryopreservation in liquid nitrogen in an appropriate freezing medium.

Step 2) is a step of activating natural killer cells, preferably 10 to 100 ng / ml, more preferably 20 to 30 ng / ml, most preferably 25 ng / ml of IL-15 On the second day of culturing isolated splenocytes, it is preferable to add IL-15. Preferably, IL-15 may be added to splenocytes and cultured for 6 days.

Step 3) is a step of separating CD5 ⁻ monocytes from splenocytes cultured in step 2), using CD5 ⁺ magnetic beads to separate only CD5 ⁻ monocytes.

Natural killer cells of the present invention

1) separating monocytes from peripheral blood of a mammal, including human;

2) separating CD56 ⁺ monocytes from isolated peripheral blood monocytes; And

3) culturing for 2 to 30 days by adding IL-15 to the isolated CD56 ⁺ monocytes

It can also manufacture by the method containing.

In more detail, the step 1) is a step of separating monocytes from peripheral blood of a healthy donor or cancer patient, using a blood sample obtained from a human body, or obtaining a monocyte concentrate by using apheresis. Density-gradient centrifugation separates only monocytes, and the isolated monocytes can be frozen and stored until later use in natural killer cell culture (Fujiwara S et al., Journal of Immunol Methods 24:90). (2); 265-273, 1986.

Step 2) is the step of separating the CD56 ⁺ monocytes for natural killer cell culture from the peripheral blood monocytes isolated in step 1), using CD56 ⁺ magnetic beads to separate only CD56 ⁺ monocytes in monocytes.

Step 3) is a step of activating natural killer cells by adding IL-15 to the isolated CD56 ⁺ monocytes and culturing, preferably 10 to 100 ng / ml, more preferably 20 to 30 ng / ml, most Preferably, 25 ng / ml of IL-15 may be used, and cultured for 14 days or more, IL-15 may be added every 3-4 days.

As a result of examining the tumor healing effect of the natural killer cells prepared as described above, the natural killer cells of the present invention showed high tumor killing ability in neuroblastoma, melanoma and the like (see FIGS. 8, 9 and 15b ).

From the above results, the natural killer cells of the present invention can induce a strong solid tumor specific cytotoxic immune response when administered to a mammal, and thus can be useful as a composition for treating solid tumors such as neuroblastoma and melanoma.

According to this aspect of the present invention, the composition for treating solid tumors using natural killer cells as an active ingredient can be administered to a target tumor by an appropriate route, and it is preferable to inject adjacent to the tumor site.

Compositions of the present invention comprise natural killer cells of the present invention in combination with one or more pharmaceutically acceptable carriers or adjuvants, which carriers or adjuvants in themselves do not cause any adverse reactions to the individual receiving the composition or It is preferred to be an adjuvant.

The dosage of natural killer cells or compositions of the present invention depends on the health and physical condition of the individual to be treated, the degree of protection desired, the assessment of the medical situation and other relevant factors. This amount is expected to fall within a relatively broad range that can be measured through routine trials, for example, from 1.26 × 10 ⁸ to 1.32 × 10 ⁹ cells per dose, based on 60 kg adult, for the treatment of neuroblastoma. It may be administered in an amount.

Another aspect of the invention relates to the use of natural killer cells of the invention in connection with the treatment of a disease state. Examples of diseases that can be treated in accordance with the methods of the invention include solid cancers such as neuroblastoma or melanoma.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Construction of Mouse NK Cells, Tumor Cell Lines, and Mouse Tumor Models

<1-1> Preparation of Mouse NK Cells

Spleens were aseptically extracted from 6-week-old A / J, C57BL / 6 and BALB / c mice purchased from SLC, Japan, to prepare single cell suspensions using mesh and pestles.

Cells were washed with lx HBSS (Mediatech, Inc) and cell down at 20 ° C., 1200 rpm for 6 minutes, then the supernatant was removed and red blood cells were removed with 2 ml ammonium chloride solution.

Wash twice with 1x HBSS and then pass through a 70 μm cell strainer (BD Falcon) in an amount of 2 ml per well to give 5 × 10 ⁶ cells / ml to each well of a 6-well plate. And cytokine IL-15 (R & D systems), IL-2 (Calbiochem), IL-12 (R & D systems) and IL-21 (R & D systems) and 1 ml of RPMI medium (Gibco, 10%) on day 2 of culture. FBS, 0.055 mM 2-mercaptoethanol, 0.1 mM sodium pyruvate, antibiotics, 2 mM L-glutamine) were added.

After further incubation for 4 days, CD5 ⁺ cells were removed using CD5 ⁺ magnetic beads (Miltenyi Biotec). Specifically, after recovering the splenocytes cultured for a total of 6 days, the pellet was put into 80 μl of MACS running buffer (PBS, 2mM EDTA, 0.5% BSA) per 1x10 ⁷ cells to release the pellet, 1x10 20 μl of CD5 ⁺ magnetic beads per ⁷ cells were added and reacted at 4 ° C. for 15 minutes. After washing with 35 ml of MACS running buffer, pellets were released with 1 ml of MACS running buffer and CD5 ^- cells were recovered using an AutoMACS cell sorter (AutoMACS cell sorter, Miltenyi Biotec).

<1-2> Preparation of Tumor Cell Line

Tumor cell lines Neuro-2a, CT26, YAC-1, EL-4, and B16F10 (ATCC) were recovered by pipetting or trypsin-EDTA, centrifuged at 1200 rpm for 5 minutes, and the supernatant removed as much as possible. It was.

25 μl of FBS and 100 μl of chromium ( ⁵¹ Cr, Sodium Chromate) solution per 1 × 10 ⁶ cells were incubated at 37 ° C. for 1 hour. Washed three times with 10 ml RPMI-1640 medium and aliquoted into each well of a 96-well plate at 1 × 10 ⁴ cells per 100 μl.

<1-3> Construction of mouse tumor model

Tumor cells (neuroblastoma, melanoma cells (ATCC)) incubated at 100 ° C. (Nunc, Denmark) at 37 ° C. and 5% CO ₂ conditions were washed with 1 × PBS (Cambrex) and trypsin-EDTA. Tumor cells were recovered. Cells were centrifuged for 6 minutes at 1200 rpm and 20 ° C, washed three times with 1X PBS, and then suspended in 1X PBS. The mice were then injected subcutaneously or intraperitoneally with an amount of 2 × 10 ⁵ cells (melanoma cell line) or 3 × 10 ⁵ cells (neuroblastoma cell line) per mouse using a 30 gauge insulin syringe.

Three days after tumor cell introduction, tumor burden was observed and randomly grouped 6-7 per group.

Example 2: Preparation of Human NK Cells

First, 100 ml of blood was collected from healthy adult men and women who did not have special diseases, and peripheral blood mononuclear cells (PBMC) were collected using Ficoll-Histopaque density gradient method (Sigma, St. Louis, Mo. ).

The isolated PBMC was reacted for 15 minutes at 4 ° C. by magnetic bead separation (AutoMACS, Miltenyi Biotec) to separate NK cells.

The isolated NK cells were diluted in RPMI1640 medium containing 10% FBS in an amount of 1.5 × 10 ⁶ cells / ml and aliquoted into 2 wells of each well of a 12-well plate.

The cytokines IL-2 and IL-15 were then treated at 3-4 day intervals.

Example 3: Evaluation of NK Cell Content and Cytotoxicity According to Kinds and Amounts of Cytokines

In order to evaluate the proliferation and killing ability of NK cells cultured by adding various cytokines as described in Example <1-1>, target cells labeled with NK cells and chromium ( ⁵¹ Cr, Sodium Chromate) (Example Tumor cell lines prepared in <1-2> (E: T = 20: 1, 10: 1, 5: 1) were injected into each well of a 96-well plate and incubated at 37 ° C. for 4 hours. After incubation, centrifuge at 2000 rpm for 3 minutes, take 100 µl of the supernatant of each well, and divide into 5 ml of green tube, and the amount of chromium released from the cancer cells by killing the cancer cells per minute at the gamma counter (γ-counter). It was measured by count number (cpm). Cancer cell killing ability (lysis (%)) was calculated by the following formula.

Lysis (%) = (ER-SR / MR-SR) x 100

In this formula, experimental release (ER) is the number of counts per minute of supernatant released from the experimental group, and spontaneous release (SR) is the amount of supernatant naturally released in the target cell control group (RPMI-1640 medium) that does not contain NK cells. Counts per minute, MR (maximum release) is the number of counts per minute of supernatant that release 100% radioactivity by adding Triton X-100 4% solution to target cells.

As a result, the addition of IL-2 increases the overall cell number, but the tumor cell killing ability is low. The addition of IL-12 and IL-21 increases the killing ability, but the total number of NK cells that can be obtained is very limited. On the other hand, when IL-15 was added, the total number of NK cells as well as tumor cell killing ability were excellent ( FIGS. 1 and 2 ). In particular, the most effective proliferation was shown when the concentration of IL-15 at 25 ng / ml was added ( FIGS. 3A and 3B ).

Example 4 Evaluation of Cytotoxicity According to Time of Addition of IL-15 (1)

NK cells can be positively selected using NK1.1, DX5 bound magnetic beads, well known as NK cell markers, or negatively selected using CD3, CD5 bound magnetic beads. It may be. In this example, NK cells were isolated by the former method.

Specifically, when NK cells are isolated and cultured in splenocytes, NK cell population can be seen to be well maintained in high purity after 6 days, but the cell killing measured in the same manner as in Example 3 The capacity was very low ( FIG . 4C ). On the other hand, NK cells isolated after culturing splenocytes for 6 days as in Example <1-1> showed very high cell killing ability while maintaining relatively high purity NK cells ( FIG. 4B) . .

Example 5 Evaluation of Cytotoxicity According to Time of Addition of IL-15 (2)

In this example, the NK cells were cultured in the same manner as in FIG. 4B, but the experiment was performed by varying the addition time of IL-15 on the second and fourth days of culture. As a result, the conditions added on day 2 were found to be more efficient in terms of increasing the number of NK cells ( FIG. 5A ).

In addition, the cytotoxicity of the tumor cell lines Neuro-2a, B16F10 and CT26 was increased by 10-15% when IL-15 was added on the second day of culture ( FIG. 5B ).

Example 6: Evaluation of Purity of NK Cells

Mouse splenocytes were cultured for 6 days using IL-15 (25 ng / ml) according to the method of Example <1-1>, and only CD5 ^- cells were finally isolated using CD5 ⁺ magnetic beads. The results are shown in FIG .

6, the culture until NK cells was only ^3-5% was increased by a ^{76% (CD3 DX5 +),} T cells (CD3 ⁺ DX5 ^-) are then incubated in culture before 33% 13% It was reduced by about half, and finally 70-88.5% of NK cells (CD3 ^- DX5 ⁺ ) were obtained. This NK cell can be selectively amplified, compared to the method of (B) in FIG. 4, CD5 than DX5 ⁺ separation method according to the process of the present ^invention can be seen that the separation method is more preferable.

Example 7: Cytotoxicity Comparison of Autologous NK Cells and Allogenic NK Cells

If the MHC and the target cell (receptor) of the target (target cell) does not match, it is recognized as a non-magnetic cell, NK cells can kill the target cells better.

In order to confirm this, in this embodiment, in vitro ( in In vitro ) NK cells were evaluated for cytotoxicity. The results are shown in Figures 7a and 7b .

As shown in FIGS . 7A and 7B , tumor cell killing ability in C57BL / 6 mice with Mb-1 type K ^b is shown in the order of neuro-2a (K ^a ), CT26 (K ^d ), and B16F10 (K ^b ). In BALB / c mice of type K ^d , neuro-2a (K ^a ), B16F10 (K ^b ), and CT26 (K ^d ) were shown. These results show that NK cells are more effective at killing allogeneic target cells than autologous target cells.

Example 8: Cytotoxicity Assessment in Mouse Tumor Models

In this example, the tumor regression was evaluated by measuring the tumor volume using a real tumor model. Tumor volume was calculated according to the following formula, which is a general formula for calculating tumor volume:

(short length) ² x (long length) x 0.523

(Here, short length means the shortest axis length of the formed tumor and long length means the longest axis length of the tumor.)

The mouse neuroblastoma and melanoma models prepared in Example <1-3> were injected with NK cells in an amount of 3 × 10 ⁶ every day for six consecutive days from the third day of tumor inoculation. The results are shown in FIGS. 8 and 9 .

8 and 9 , in the neuroblastoma model, the autologous NK cell introduction group showed 80% tumor regression compared to the PBS introduction group, while the homologous NK cell introduction group showed 90% tumor regression. In the melanoma model, both autologous and allogeneic NK cell introduction groups showed similar levels of tumor regression and mouse survival.

Example 9 Changes of T Cells and NK Cells in a Mouse Neuroblastoma Model Treated with NK Cells

In the mouse neuroblastoma model, the tumor size was tracked after 6 times of NK therapy, and the anti-cancer effects of NK cells were confirmed.The immune cells in the spleen and lymph nodes of the treated and untreated mice were treated on the 28th day. ) Was confirmed by FACS (fluorescence activated cell sorter). The results are shown in FIGS. 10A, 10B and 11 .

As shown in FIGS. 10A and 10B , the PBS control group had a lower level of CTL cells (CD3 ⁺ CD8 ⁺ ) and Th cells (CD3 ⁺ CD4 ⁺ ) in the spleen and lymph nodes than the normal mouse group without tumor injection. However, when comparing mice treated with NK cells with those not treated, the levels of CTL and Th cells were higher than normal in treated mice, indicating that effective T cells were activated ( FIG. (A), (B), (C) and (D) of 10a . However, no changes in NK cells (CD3 ^- DX5 ⁺ ) were observed in the spleen and lymph nodes ( (E) and (F) in FIG. 10B ). These results show that tumors can be treated through the proliferation and activation of CTL by NK cells.

In particular, after administration of CD5 ^- NK cells, the increase in activated CTL and memory CTL in the spleen was confirmed ( FIG. 11 ).

Example 10 Content and Activation Degree of NK Cells in Human PBMC

PBMCs were isolated from blood of normal individuals as described in Example 2.

PBMCs isolated after blood collection contained about 10-20% of NK cells ( FIG. 12A ), but NKp44, an NK cell activation marker, was hardly expressed ( FIG. 12B ).

Example 11 Evaluation of Tumor Cell Killing Capacity of Human NK Cells

CD56 ⁺ cells isolated from PBMC were treated with IL-15 (50 ng / ml) for 14 days as described in Example 2, and the killing ability of tumor cell lines was compared using the cultured human NK cells. .

As a result, NK cells ( FIG. 13A ) isolated using CD56 ⁺ magnetic beads showed high levels of tumor killing ability, whereas T cells ( FIG. 13B ) showed very low killing ability.

Example 12 Assessment of Tumor Cell Killing Capacity According to the Mismatching Level of Blood Provider's KIR Type and HLA Type of Tumor Cells

NK cells do not recognize and kill themselves when they express the same MHC molecule on the surface of the cell, but can directly kill when they express tumor cells or other MHCs with low MHC expression.

Thus, in order to evaluate the killing ability of the target cell according to the difference between the blood donor's KIR type and tumor cell's HLA type, NK cells were provided with blood from two blood donors with different KIR types. After 14 days of incubation, tumor cell killing ability was compared according to the extent of mismatch with HLA.

As a result, it was shown that tumor cell killing ability was better when only one or two KIR matched than when the KIR type of the blood donor and the HLA type of the tumor cell matched completely ( FIGS. 14A and 14B ).

Example 13: Evaluation of proliferation rate and tumor cell killing ability according to the isolation of human NK cells

Human NK cells were isolated and cultured for about 3 weeks (21 days) according to the method of Example 2 to compare the proliferation rate and cell killing ability.

As a result, in the total number of cells, culturing without separation (about 11-fold increase) was higher than that of cultivating and cultivating (about 3-fold increase) ( FIG. 15A ), but the killing ability to actual tumor cells was separated. The cultured NK cells were found to be 20-30% higher ( FIG. 15B ).

Figure 1 shows the change in the total number of cells and NK cells when cultured mouse splenocytes using various combinations of cytokines (A: total cell number, B: NK cell number);

Figure 2 shows the killing of tumor cells when cultured mouse splenocytes using various combinations of cytokines (YAC-1: mouse lymphoma cells (positive control), EL-4: white leukemia cells (negative control) Neuro-2a: neuroblastoma cell line, B16F10: melanoma cell line, CT26: colorectal carcinoma cell line);

3A and 3B show the best results of NK cell activation when mouse splenocytes were cultured using 25 ng / ml IL-15;

Figure 4 shows the degree of cytotoxicity when cultured for 6 days after treatment with IL-15 in splenocytes and when treated with IL-15 after separating DX5 ⁺ NK cells from splenocytes (A : Bulk NK cell culture, B: DX5 ⁺ NK cells isolated after culturing, C: NK cells isolated after culturing with DX5 ⁺ );

FIG. 5 shows the cytotoxicity of the isolated DX5 ⁺ NK cells after incubation with IL-15 on days 2 and 4 of splenocytes cultured (Neuro-2a: neuroblastoma cell line, B16F10: black color) Species cell line, CT26: colorectal carcinoma cell line);

6 is a comparison of the purity of bulk NK cells cultured using IL-15 for 6 days and CD5 ^- NK cells isolated using CD5 ⁺ magnetic beads;

7a and 7b show the comparison of the activity and cytotoxicity of allogeneic NK cells and autologous NK cells (Neuro-2a: neuroblastoma cell line, B16F10: melanoma cell line, CT26: colorectal carcinoma cell line);

8 shows the extent of tumor regression by CD5 ^- NK cells in a mouse neuroblastoma model;

9 shows the extent of tumor regression by CD5 ^- NK cells in a mouse melanoma model;

10A and 10B show changes in T cells and NK cells in the spleen and lymph nodes after treatment of CD5 ^- NK cells in a mouse neuroblastoma model;

Figure 11 shows the comparison of the contents of activated CTL (A), Th cells (B), memory CTL (C) and memory Th cells (D) in the spleen after treatment of mouse neuroblastoma model CD5 ^- NK cells ;

12 shows the content and activation of NK cells in human PBMC;

13A and 13B show tumor cell killing ability of NK cells obtained by incubating 14 days of treatment with IL-15 in CD56 ⁺ cells isolated from human PBMC according to the method of the present invention ( FIG. 13A : Purified CD56 ⁺ NK Cells, FIG. 13B : CD3 ⁺ T cells, K562: thymoma cell line, SK-N-SH: neuroblastoma cell line with matching KIR type of blood donor and 2 HLA, SK-N-BE (2): KIR of blood donor Neuroblastoma cell line, which perfectly matches the type, SK-N-DZ (G1): neuroblastoma cell line in which one HLA matches the KIR type of the blood donor);

14A and 14B show tumor cell killing ability according to the difference between the HIR type of the blood donor and the KIR type of the blood donor (green bar: NK cell, yellow bar: T cell, K562: thymic cell line, SK-N-SH) : Neuroblastoma cell line with KIR type matched with two HLA of blood donor, SK-N-BE (2): Neuroblastoma cell line with KIR type matched with blood donor, SK-N-DZ (G1): Blood donor Neuroblastoma cell line with a KIR type of 1 and one HLA);

15A and 15B show NK cells cultured for 21 days by treating IL-15 on CDB ⁺ cells isolated from human PBMCs and PBMCs obtained by culturing NK cells for 21 days by treating IL-15 in human PBMCs. Cell proliferation ( FIG. 15A ) and tumor cell killing ability ( FIG. 15B ) for the cells.

Claims (14)

delete delete delete delete delete 1) separating monocytes from peripheral blood of a mammal, including human; 2) from the separated peripheral blood mononuclear cells and separating the CD56 ⁺ monocytes only processes using CD56 ⁺ magnetic beads; And 3) culturing for 2 to 30 days by adding IL-15 to the isolated CD56 ⁺ monocytes Containing, method for producing natural killer cells. The method of claim 6, In step 3), the method for producing natural killer cells, characterized in that the IL-15 is added in an amount of 10 to 100 ng / ml. Claim 8 was abandoned when the registration fee was paid. The method of claim 6, In step 3), the method for producing natural killer cells, characterized in that the addition of IL-15 in an amount of 20 to 30 ng / ml. Claim 9 was abandoned upon payment of a set-up fee. The method of claim 6, In step 3), the production method of natural killer cells, characterized in that the addition of IL-15 in an amount of 25 ng / ml. The method of claim 6, In step 3), the method for producing natural killer cells, characterized in that culture for 14 to 30 days by adding IL-15 to the isolated CD56 ⁺ monocytes. A natural killer cell prepared by the method of claim 6 and capable of inducing a solid tumor specific immune response. The method of claim 11, Natural killer cells, wherein the natural killer cells are allogenic natural killer cells. A composition for treating solid tumors, comprising the natural killer cells of claim 11 as an active ingredient and comprising one or more pharmaceutically acceptable carriers or adjuvants. The method of claim 13, A composition characterized in that the solid tumor is melanoma, neuroblastoma or colorectal carcinoma.

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