EA038848B1 - Method for proliferating natural killer cells and compositions for proliferating natural killer cells - Google Patents

Abstract

The invention relates to a method for proliferating natural killer cells and a composition for proliferating natural killer cells, capable of improving a cell proliferation effect and an anticancer effect, wherein the method comprises a step for culturing natural killer cells in a culture liquid containing a first interleukin (IL-2), a second interleukin (at least one interleukin selected from the group consisting of IL-12, IL-15, and IL-18), and an anti-NKp46 antibody.

Description

Technology area

The present disclosure relates to a method for proliferating natural killer cells, a composition for proliferating natural killer cells, and an enhanced anti-cancer effect of proliferating natural killer cells.

Background of the invention

Natural killer cells (hereinafter NK cells) used in cellular immunotherapy are cells that are morphologically characterized by large cytoplasmic granules and which make up about 5 to 15% of the lymphocyte content in the blood. The main functions of NK cells identified to date include the ability to kill tumor cells, cytotoxicity against virus-infected cells, the ability to kill bacteria and fungi, etc. Therefore, NK cells are expected to play an important role in anti-tumor immunity and protective immunity against microorganisms.

NK cells are classified into a separate class of immune cells, different from T cells or B cells, because they do not have receptors on the cell surface, such as the T cell receptor, CD4, or immunoglobulin. In particular, NK cells are known to be capable of killing tumors nonspecifically. This ability of NK cells is used in the treatment of solid tumors together with lymphokine-activated killer cells (LAK) and tumor-infiltrating lymphocytes (TIL) or for immunotherapy by infusion of donor lymphocytes (Tilden. A.V. et al., J. Immunol., 136 ), which is used as a modern cell therapy to prevent rejection in bone marrow transplants or organ transplants. It has also been reported that impaired differentiation or activity of NK cells is associated with various cancers, including breast cancer (Konjevic G, et al., Breast Cancer Res. Treat. 66: 255-263, 2001), melanoma (Ryuke Y, et al., Melanoma Res., 13: 349-356, 2003), lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002), etc., and therefore NK cell therapy appeared in the field of treatment of these diseases.

For the effective use of NK cells in cellular immunotherapy, such as the treatment of cancer, a large number of NK cells are required. However, NK cells make up only 5 to 15% of the lymphocyte count in the blood of a healthy person, as described above. In particular, the number, differentiation, and functioning of NK cells are often reduced in cancer patients, and it is indeed difficult to provide a sufficient number of NK cells to treat diseases. In addition, NK cells created by existing methods of NK cell proliferation differ from each other in proliferation rate and cytotoxicity. Therefore, there is an urgent need for a new method for the proliferation or differentiation of NK cells that would provide a large number of NK cells that retain the ability to kill cancer cells at a sufficient level.

Detailed description of the invention

Technical problem

The object of the present disclosure is to provide a method for the proliferation of natural killer cells with improved antitumor activity.

Another object of the present disclosure is to provide a method for producing natural killer cells.

Another object of the present disclosure is to provide a pharmaceutical composition for the treatment or prevention of cancer, comprising natural killer cells.

It is yet another object of the present disclosure to provide a composition for the proliferation of natural killer cells.

Technical solution

In one aspect, there is provided a method for proliferating natural killer cells (NK cells), the method comprising culturing NK cells in a culture medium comprising a first interleukin, a second interleukin, and an anti-NKp46 antibody.

The first interleukin may be IL-2.

The second interleukin can be one or more interleukins from the group consisting of interleukin-12 (IL-12), interleukin-15 (IL-15) and interleukin-18 (IL-18). Preferably, the second interleukin is IL-12, IL-18, IL-12 / IL-18, or IL-12 / IL-15 / IL-18, and more preferably IL-18.

Culturing NK cells can be culturing cells in a culture medium containing a first interleukin, a second interleukin, and an anti-NKp46 antibody in the presence of gammaglobulin (IgG) and fibronectin. The method of the present disclosure can further improve the anti-cancer effect of NK cells by culturing NK cells in the presence of gamma globulin (IgG) and fibronectin.

In this context, the term cell proliferation or cell proliferation means that the number of cells increases in the culture medium during sequential cell division or differentiation of cells. In this context, the term NK cell proliferation means that the number of NK cells increases in the culture medium during sequential cell division, or the number of NK cells increases.

- 1 038848 due to the differentiation of immature blood cells into NK cells. Therefore, the proliferation of NK cells can include an increase in the number of NK cells, the differentiation of undifferentiated lymphocytes into

NK cells by acquiring the traits of NK cells or maturation of NK cells from immature NK cells.

The NK cell, which is the starting material for proliferation, can be purchased commercially or obtained from a human or animal, preferably a human, in need of NK cell therapy. In addition, the NK cell can be isolated from any tissue source in the body. For example, NK cells can be found in blood taken from the body. Provided that the blood contains NK cells, it can be used as a source of NK cells for carrying out the NK cell proliferation method, for example, it can be whole blood, umbilical cord blood, bone marrow or peripheral blood.

Proliferation of NK cells may include, for example, culturing peripheral blood mononuclear cells. The term peripheral blood mononuclear cells (PBMC) means mononuclear cells isolated from the peripheral blood of a mammal, preferably a human, and includes immune cells such as B cells, T cells and NK cells, and granulocytes such as basophils, eosinophils and neutrophils. ... PBMC can be obtained from peripheral blood taken from a living organism in accordance with the general method of obtaining. Preferably, PBMC can be isolated from peripheral blood by specific gravity centrifugation using Ficoll.

PBMC can be obtained from a subject in need of treatment, namely, it can be an autologous mononuclear peripheral blood cell. If PBMC is an autologous mononuclear peripheral blood cell, then this is preferable, since in this case it is not necessary to remove T cells, because all cells are from the patient himself, although some T cells are present in proliferating NK cell populations.

PBMCs used in the NK cell proliferation method of the present invention can be cryopreserved. In a specific embodiment, after the PBMCs are isolated from the blood, they can be frozen. The PBMC can then be thawed and used to proliferate NK cells. Freezing can be performed by a method known in the art, preferably by a method comprising a first freezing in a temperature range of 4 ° C to -42 ° C, a second freezing in a temperature range of -42 ° C to -15 ° C, and a third freezing in a temperature range from -15 ° C to -120 ° C. In this case, the temperature at each freezing can be raised or lowered to the upper limit or the lower limit of the temperature range of each freeze by holding the sample for a predetermined time sequentially at alternating temperatures within the temperature range. In a specific embodiment, the first freezing can be performed at 0 ° C for 10-15 minutes, at -12 ° C for 5-10 minutes, at -42 ° C for 0.5-1 minutes. After the first freeze, the second freeze can be performed at -25 ° C for 13 minutes and at -15 ° C for 1-3 minutes, and after the second freeze, the third freeze can be performed at -42 ° C for 20-40 minutes and at -120 ° C for 20-50 min. In another embodiment, the first freezing can be performed in the temperature range from 4 to -40 ° C at a rate of 0.5 to 5 ° C / min, the second freezing can be performed in the temperature range from -40 to -90 ° C at a rate of 1 up to 10 ° C / min after the first freezing, and the third freezing can be performed in the temperature range from -90 to -120 ° C at a rate of from 1 to 10 ° C / min after the second freezing. Each freezing of the freezing method can be performed using a programmable cryo-freezer (CRF -controlled rate freezer). In freezing the PBMCs of the present invention, the PBMCs are preferably frozen after adjusting the number of isolated cells to the desired value by flotation of the cells in a mixture of CryoStor CS10 or Aly505NK-EX and albumin + DMSO. After thawing the cells, frozen PBMCs can be used to culture and proliferate NK cells in accordance with the present invention, and the freezing method has the advantages that NK cells are usable by culture and proliferation processes at any time that NK cells are required after blood is drawn from the patient. The disadvantage of most cellular immunotherapies is that blood must be drawn from the patient every two weeks, taking into account the culture period with a specified 2-week incubation period, in order to obtain a clinically effective number of cells, and the patient's condition can affect the cell culture. However, the present invention can provide a method for freezing and thawing PBMCs that can maintain a high survival rate and can be successfully used in a method for culturing activated NK cells. Thus, it allows NK cells to be obtained regularly and administered to a patient regardless of the patient's condition, since a large volume of the patient's blood can be drawn, separated and frozen / stored when the patient is in good condition.

A method for proliferating NK cells according to one embodiment may include culturing the NK cells in the presence of gamma globulin (IgG) and fibronectin. Culturing in the presence of gamma globulin (IgG) and fibronectin can be, for example, culturing cells in a culture medium containing gamma globulin and fibronectin, or culturing cells in a culture plate in which the culture surface (the surface of the culture plate extending in contact with cells) is coated with gamma globulin (IgG) and fibronectin. It is preferred that the cultivation of NK cells can be carried out in a culture plate coated with gammaglobulin and fibronectin. A culture plate coated with gamma globulin and fibronectin can be prepared by applying a solution containing gamma globulin and fibronectin to the plate. Instead of fibronectin, a known adhesive protein, collagen, for example, can be used. In one example, a gamma globulin and fibronectin coating can be applied to a culture plate by adding a solution containing gamma globulin and fibronectin to a culture plate (eg, a T75 flask) and incubating the culture plate at low temperature.

The concentration of gamma globulin can be from 0.1 to 1 ng / ml, from 1 to 10 ng / ml, from 10 to 100 ng / ml, or from 1 to 100 ng / ml. Gamma globulin can activate NK cells by stimulating the FcyRIII receptor on NK cells.

The concentration of fibronectin can be from 0.1 to 50 μg / ml, from 1 to 50 μg / ml, from 5 to 50 μg / ml, or from 10 to 50 μg / ml. Fibronectin can facilitate cell-to-cell communication by promoting NK cell movement.

A method for proliferating NK cells according to one embodiment can be carried out by culturing NK cells in a culture medium containing a first interleukin (IL-2), a second interleukin (one or more interleukins selected from the group consisting of IL-12, IL-15 and IL18) and anti-NKp46 antibody. The culture medium can be one obtained by introducing an anti-NKp46 antibody, a second interleukin and IL-2 into a normal immune cell medium. The environment for immune cells can include, for example, ALyS505NK-EX (Cell Science & Technology Inst. Inc., Japan), RPMI1640 (Life technologies, USA), x-vivo10 (Lonza, USA), CellGro SCGM (CellGenix, Germany), KBM (Kohjin bio, Japan), etc. A culture medium according to one embodiment of the present invention can be prepared by introducing an anti-NKp46 antibody, a second interleukin and IL-2 into the immune cell medium described above. A culture medium according to one embodiment of the present invention can also be prepared by administering an anti-NKp46 antibody and a second interleukin to a commercially available immune cell medium that already contains IL-2.

The culture medium can, without limitation, be any immune cell culture medium, provided that the immune cell medium contains a second interleukin, an anti-NKp46 antibody, and IL-2.

The concentration of IL-2 in the culture medium can be from 500 to 5000 IU / ml, from 600 to 4000 IU / ml, from 700 to 3000 IU / ml, from 800 to 2000 IU / ml, from 900 to 1500 IU / ml, from 900 to 1200 IU / ml, or 1000 to 1200 IU / ml. IL-2 can facilitate the growth of NK cells.

The concentration of anti-NKp46 antibody in the culture medium can be from 0.1 to 10 μg / ml, from 0.5 to 10 μg / ml, from 1 to 10 μg / ml, or from 5 to 10 μg / ml. NKp46 is an activating receptor for NK cells, and thus the NKp46 antibody of the present invention can activate NK cells by stimulating NKp46.

The concentration of the second interleukin in the culture medium can be from 0.1 to 100 ng / ml, from 1 to 100 ng / ml, from 10 to 100 ng / ml, from 20 to 100 ng / ml, from 30 to 100 ng / ml, 50 to 100 ng / ml or 70 to 100 ng / ml. The second interleukin of the present invention is a component that is able to increase the proliferation efficiency of NK cells and greatly improve the activation of NK cells. Therefore, the use of a second interleukin in accordance with the present invention in addition to IL-2 and an anti-NKp46 antibody can demonstrate an enhancing and synergistic effect on NK cell proliferation and activation by IL-2 and anti-NKp46 antibodies, and, accordingly, the present invention can provide improved effect on NK cell proliferation compared to the known method for NK cell proliferation.

As a result, the combination of IL-2, a second interleukin, and an anti-NKp46 antibody, which are components of the NK cell proliferation composition of the present invention, can maximize the proliferation and culture efficiency of NK cells, and the present invention is very economical due to the use of a combination of only main components.

The culture medium may further comprise plasma or serum. Plasma or serum included in the culture medium can be obtained from the peripheral blood from which the PBMCs used for the proliferation of NK cells were isolated. The plasma concentration can be from 1 to 20 v / v%, from 1 to 15 v / v%, from 2 to 15 v / v%, from 5 to 15 v / v% or from 5 up to 10 v / v% in relation to the total volume of the culture medium.

In a specific embodiment, PBMCs and plasma (or serum) can be isolated from the peripheral blood of a patient to be injected with NK cells, respectively, and the resulting PBMCs can be cultured in a culture medium containing IL-2, a second interleukin, an anti-NKp46 antibody, and plasma ( or serum) isolated from peripheral blood. It is preferable to carry out the culture in a culture plate coated with gamma globulin and fibronectin.

Culturing PBMCs can be carried out under conventional cell culture conditions, i. E. in a CO ₂ incubator at 37 ° C, and it can be continued with the addition of culture medium every 2 or 3 days. The concentration of PBMCs introduced into the culture medium can be from 4x105 to 5x10 ⁷ cells / ml, but not

- 3 038848 is subject to these terms and conditions. The cultivation period can last, for example, 7 to 20 days, 8 to 18 days, 10 to 16 days, 10 to 15 days, or 10 to 14 days, but is not limited thereto. To ensure the required number of NK cells, the cultivation period can be determined, respectively, within the above range or outside the above range. The culture plate can be a dish, flask, plate, multi-well plate and culture bag, which are commercially available.

Meanwhile, in order to maximize the proliferation yield of NK cells, the cultivation can be carried out in stages. In this case, the components of the culture medium, the culture plate and the culture period at each stage may be the same or different from each other. By finding the optimal culture period for each stage, NK cell proliferation can ultimately be achieved. In addition, the present invention is characterized in that the cultivation is performed by introducing the cultivation composition (or the culture medium) into the flask at an interval of 2-3 days during the cultivation period, thereby maintaining the NK cell culture.

In one embodiment, the method for proliferating NK cells of the present invention may comprise first culturing PBMC culture by introducing PBMC and a culture medium comprising a first interleukin, a second interleukin, an anti-NKp46 antibody and plasma into a culture plate coated with gamma globulin and fibronectin; and a second culturing culturing the culture obtained in the first culture by further introducing there a culture medium comprising a first interleukin, a second interleukin, an anti-NKp46 antibody and plasma. In the proliferation method, the first interleukin may be interleukin-2 (IL-2). Further, the second interleukin may be one or more interleukins from the group consisting of interleukin-12 (IL-12), interleukin-15 (IL-15) and interleukin-18 (IL-18). The first interleukin and the second interleukin are the same as described above.

Plasma can be obtained from peripheral blood from which the PBMCs were obtained. Serum can be used instead of plasma.

The concentration of IL-2 in the culture medium during the first cultivation can be from 500 to 5000 IU / ml, from 600 to 4000 IU / ml, from 700 to 3000 IU / ml, from 800 to 2000 IU / ml, from 900 to 1500 IU / ml, from 900 to 1200 IU / ml or from 1000 to 1200 IU / ml.

In the first culture, PBMC can be cultured for 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, for example 3, 4, 5, 6 days, preferably 5 days, in a culture medium containing the first interleukin (IL-2), a second interleukin (one or more interleukins selected from the group consisting of IL-12, IL-15 and IL-18), an anti-NKp46 antibody and plasma.

The method for proliferating NK cells according to the present invention may include the subsequent introduction of a culture medium comprising IL-2 and plasma into the culture during the first culture. For example, a culture medium comprising IL-2 and plasma for subsequent administration can be administered on day 3 and / or day 4 after the first culture. The concentration of IL-2 in the culture medium for subsequent administration may be the same as in the culture medium in the first culture, but is not limited thereto. The volume of the culture medium intended for subsequent administration may be equal to the volume of the culture.

In a specific embodiment, the NK cell proliferation method of the present invention comprises a second cultivation of cultivation of the culture obtained in the first cultivation by additionally introducing there a culture medium containing a first interleukin (IL-2), a second interleukin (one or more interleukins selected from the group, consisting of IL-12, IL15 and IL-18), anti-NKp46 antibody and plasma.

The second culture can be performed by transferring the culture from the first culture to a new plate. The new tablet may not include gamma globulin and fibronectin. The concentration of IL-2 in the culture medium in the second culture may be the same as in the culture medium in the first culture, but is not limited thereto. The concentration of IL-2 in the culture medium during the second cultivation can be from 500 to 5000 IU / ml, from 600 to 4000 IU / ml, from 700 to 3θ0θ IU / ml, from 800 to 2000 IU / ml, from 900 to 1500 IU / ml, from 900 to 1200 IU / ml or from 1000 to 1200 IU / ml. The volume of culture medium containing the first interleukin, the second interleukin (one or more selected from the group consisting of IL-12, IL-15 and IL-18), anti-NKp46 antibody and plasma, which is introduced into the culture in the second culture, can be equal to the volume of culture.

The cells can be cultured in the culture medium in a second culture for 1, 2, 3, 4, 5 or 6 days, preferably 1, 2, or 3 days.

The NK cell proliferation method of the present invention may further comprise a third culturing culture after reducing the IL-2 concentration in the culture to 4/5 or less — to 1/10 or less relative to the concentration before the culture introduction. The IL-2 concentration can be, for example, 1/100 to 3/5, 1/100 to 1/4, 1/100 to 2/5, 1/100 to 1/3, or 1/100 to 1/5 relative to the concentration before the introduction of the culture.

The concentration of IL-2 in the culture medium can be reduced by introducing the culture medium without IL-2 into the culture. The culture medium introduced in the third culture may contain plasma.

- 4 038848

The concentration of IL-2 in the culture during the third cultivation can be from 1500 to 1300 IU / ml, from

1300 to 1000 IU / ml, 1000 to 600 IU / ml, 600 to 500 IU / ml, 500 to 400 IU / ml, 400 to

300 IU / ml, 300 to 200 IU / ml, or 200 to 100 IU / ml.

The culture medium for each cultivation may contain suitable proteins, cytokines, antibodies, compounds and other components, provided that they do not interfere with the proliferative effect of NK cells.

In a specific embodiment, the proliferated cells can express one or more selected from the group consisting of CD56, CD16, NKG2D, perforin, and granzyme B. For example, the expression can be the expression of a large number of the above factors as compared to peripheral blood cells or their populations prior to proliferation or NK cells or their populations prior to proliferation. For example, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or at least 100% of cells peripheral blood or their populations, including proliferated NK cells, or proliferated NK cells or their populations from the total number of cells or cell populations can express the above factors. And% expression or expression level means the percentage of NK cells expressing one or more of the above factors, relative to the total number of cells in the sample. Expression or level of expression of a factor in cells or cell populations can be determined by the value determined by flow cytometry.

In another aspect, the present invention provides a production method further comprising preserving NK cells proliferated by the NK cell proliferation method after suspending the NK cells in a solution containing dextran and albumin.

In one example, it was found that NK cells proliferated by the NK cell proliferation method of the present invention expressed anticancer agents in high yield and showed remarkable anticancer effects when compared to PBMC (see FIGS. 7-9).

In another aspect, there is provided a pharmaceutical composition for the treatment or prevention of cancer, comprising proliferated NK cells.

The NK cells included in the pharmaceutical composition and the NK cell proliferation method are the same as described above.

The subject can be a mammal, including a human.

The cancer can be, but is not limited to, but not limited to, leukemia, breast cancer, ovarian cancer, brain cancer, malignant melanoma, stomach cancer, liver cancer, colorectal cancer, or lung cancer.

The pharmaceutical composition may include cell populations of NK cells cultured by the NK cell proliferation method. The composition may include the culture medium used in the culture, or culture in addition to the cell populations cultured by this method. The composition may include a new culture medium or saline, not containing cell populations isolated from the culture, and additional components such as cytokines. The new culture medium can be a medium whose composition is identical or different from the composition of the medium used for cultivating NK cells. In a pharmaceutical composition, the administered dose of NK cells may vary depending on the condition and body weight of the patient, the severity of the disease, the mode of administration, the route and duration of administration, and for example, the administered dose may be from 1x106 to 1x109 cells / kg, preferably from 1x10 ⁷ to 1x109 cells / kg. The introduction can be carried out one or more times a day. When formulated as a liquid unit dosage formulation such as solution, suspension, emulsion, etc., the pharmaceutical composition may be administered to a patient at the above cell concentrations.

A pharmaceutical composition according to the present invention may comprise NK cells proliferated by the method of the present disclosure as described above, and may further comprise a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared according to a conventional method as a parenteral preparation such as a solution, suspension, emulsion, lyophilized preparation, etc. The pharmaceutically acceptable carrier may include an aqueous diluent such as phosphate buffered saline, purified water, sterile water, etc., or a solvent. The pharmaceutically acceptable carrier may include other conventional preservatives, etc. The pharmaceutical composition may include various anti-cancer agents or other therapeutic agents in addition to NK cells or NK cell populations.

In yet another aspect, there is provided a method for treating or preventing cancer, comprising administering proliferated NK cells to a subject.

The subject and the cancer are the same as described above.

When proliferated NK cells are administered, the route of administration may be oral or parenteral (eg, injection), and the injection may be intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection, but is not limited to, but not limited to.

In yet another aspect, there is provided the use of proliferated NK cells in the preparation of a therapeutic or prophylactic agent against cancer.

In yet another aspect, there is provided a composition for NK cell proliferation, comprising a first interleukin (IL-2), a second interleukin (one or more interleukins selected from the group consisting of interleukin-12 (IL-12), interleukin-15 (IL- 15) and interleukin-18 (IL-18)) and anti-NKp46 antibody.

In yet another aspect, there is provided the use of a composition comprising a first interleukin (IL2), a second interleukin (one or more interleukins selected from the group consisting of interleukin-12 (IL-12), interleukin-15 (IL-15) and interleukin-18 (IL-18)) and an anti-NKp46 antibody, in the preparation of a composition for NK cell proliferation.

The composition for NK cell proliferation may further comprise gamma globulin and fibronectin. In the composition, the first interleukin and the second interleukin are the same as described above.

The concentration of IL-2 in the composition for the proliferation of NK cells can be from 500 to 5000 IU / ml, from 600 to 4000 IU / ml, from 700 to 3000 IU / ml, from 80o to 2000 IU / ml, from 900 to 1500 IU. / ml, from 900 to 1200 IU / ml or from 1000 to 1200 IU / ml.

The concentration of the second interleukin in the composition for NK cell proliferation can be from 0.1 to 100 ng / ml, from 1 to 100 ng / ml, from 10 to 100 ng / ml, from 20 to 100 ng / ml, from 30 to 100 ng / ml, 50 to 100 ng / ml, or 70 to 100 ng / ml.

The concentration of anti-NKp46 antibody in the NK cell proliferation composition can be from 0.1 to 10 μg / ml, 0.5 to 10 μg / ml, 1 to 10 μg / ml, or 5 to 10 μg / ml.

The NK cell proliferation composition can be designed to proliferate or differentiate NK cells from PBMCs.

The composition for NK cell proliferation may further comprise plasma or serum. The plasma or serum to be incorporated into the composition can be obtained from blood from which the NK cells were isolated. In a specific embodiment, the plasma included in the NK cell proliferation composition can be obtained from peripheral blood from which the PBMCs are isolated.

The composition for the proliferation of NK cells may include the main components for the cultivation of PBMC and the proliferation of NK cells, other carriers or excipients, in addition to gamma globulin, fibronectin, the first interleukin (IL-2), the second interleukin (one or more selected from the group consisting of IL-12, IL-15 and IL-18) and anti-NKp46 antibody.

The beneficial effect of the invention

A natural killer (NK) cell proliferation method or composition for NK cell proliferation according to one aspect can promote the proliferation and differentiation of NK cells, and can stably provide NK cells with increased cytotoxicity. The use of NK cells obtained by this method can be useful in the treatment of diseases, such as the treatment of oncological diseases.

Description of graphic materials

FIG. 1 shows the results as the total number of cells (A) and the fold increase in the number of NK (natural killer) cells (B) using the proliferation method according to a particular embodiment of the present invention.

FIG. 2 shows the results as the total number of cells (A) and the percentage of NK cells (B) obtained by the proliferation methods according to various specific embodiments.

FIG. 3 shows the results of the pattern of NK cell proliferation depending on the concentration of IL-2.

FIG. 4 shows the results of NK activity versus IL-12 and IL-18 concentrations.

FIG. 5 shows the results of flow cytometry of NK cells obtained by the proliferation method according to a particular embodiment of the present invention.

FIG. 6 shows the results of flow cytometry of NK cell receptor activation obtained according to a particular embodiment.

FIG. 7 shows the results of flow cytometry in the study of the expression of anti-cancer substances in NK cells obtained according to a particular embodiment.

FIG. 8 shows the results of flow cytometry in the study of the% lysis of cancer cells of the leukemia cell line - K562 - NK cells obtained according to various specific embodiments.

FIG. 9 shows the results of flow cytometry in the study of% lysis of cancer cells K562 (chronic leukemia), OVCAR3 (ovarian cancer), Hep3B (liver cancer), HepG2 (liver cancer), A704 (kidney cancer) and DU145 (prostate cancer) by NK cells, obtained according to a specific embodiment.

FIG. 10 shows the characteristics of PBMC of Donor 1 before cultivation, where A is the percentage of NK cells (Q9, CD3-CD56 +), B is NK cells expressing CD16 (Q2, CD16 + CD56 +), C is B cells (Q5 , CD3-CD19 +) and D represents cytotoxicity

- 6 038848 in K562 cells (7AAD +, E: T = 10: 1).

FIG. 11 shows the characteristics of PBMC of Donor 1, cultured for 14 days after the freezing and thawing processes, where A is the percentage of NK cells (Q5, CD3 CD56 +), B is NK cells expressing CD16 (Q10, CD16 + CD56 +), C is B cells (Q1, CD3-CD19 +), D is cytotoxicity in K562 cells (7AAD +, E: T = 10: one).

Technical implementation of the invention

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are given for illustrative purposes only, and the scope of the present invention should not be limited by them.

Example 1. Obtaining mononuclear cells of peripheral blood (PBMC).

1. Isolation of PBMC and autologous plasma from blood.

Blood was taken from a vein of a healthy person. During this procedure, a blood collection cannula containing heparin was used. Each 30 ml of blood drawn from a patient was carefully transferred into two tubes (# 352070, BD, or equivalent, or better specification) with Ficoll (# 17-144002, GE Healthcare, or equivalent, or better specification). The tubes with blood were centrifuged for 10 min at 2500 rpm with breaking off and then each upper layer of plasma was transferred to a new tube.

The transferred plasma was inactivated for 30 min in a thermoblock and then centrifuged for 5 min at 4000 rpm. The supernatant in the centrifuged tube was transferred to another, new tube labeled plasma and stored at 2 to 8 ° C.

Plasma was collected from a tube containing blood and Ficoll after centrifugation, and the light yellow layer in the remaining bottom layer was carefully transferred to a new tube, being careful not to mix with the red blood cell layer, and then Ca / Mg-free DPBS (saline Dulbecco's Phosphate Buffered) (# 14190, Gibco). Thereafter, the tube was centrifuged for 5 min at 1500 rpm and the supernatant was then discarded. The pelleted cells remaining after discarding the supernatant were suspended in 5 ml of red blood cell lysis buffer (# 158904, Qiagen). The cell suspension was centrifuged for 5 min at 1500 rpm and the supernatant was discarded, and then Ca / Mg-free DPBS was added to the tube, followed by centrifugation for 5 min at 1500 rpm. The pelleted cells remaining after discarding the supernatant were suspended in 1 ml of Alys505NK-EX culture medium (# 01410P10, CSTI).

A small amount of cell suspension in Alys505NK-EX was diluted 100-fold in Ca / Mg-free DPBS, and then a small amount of the diluted suspension was mixed with an equal volume of trypan blue, and then the number of cells and their viability were counted using a hemocytometer.

2. Freezing PBMC.

All suspensions obtained in example 1.1 were centrifuged for 5 min at 1500 rpm and the supernatant was discarded. The cells were suspended in Cryostor CS10 or ALyS505NKEX + albumin + DMSO mixture stored at 2 to 8 ° C so that the cell count was 1x106 to 100x106 cells / ml. Each 1 ml of cell suspensions was separated into a 2 ml cryogenic vial, and the first freezing was performed at 0 ° C for a period of 10 to 15 min, at 12 ° C for a period of 5 to 10 min, and at -42 ° C for period from 0.5 to 1 min, using CRF (programmable cryofreezers). After the first freezing, a second freezing was performed at -25 ° C for a period of 1 to 3 minutes and at -15 ° C for a period of 1 to 3 minutes. After the second freezing, freezing was performed at -42 ° C for a period of 20 to 40 minutes and at -120 ° C for a period of 20 to 50 minutes. Alternatively, the first freezing was performed at a speed of 3 ° C / min in the temperature range from 4 to -40 ° C, the second freezing was performed at a speed of 5 ° C / min in the temperature range from -40 to -90 ° C after the first freezing and an additional freezing was performed at a rate of 5 ° C / min in the temperature range from -90 to -120 ° C after the second freezing. The frozen cells were transferred to an LN ₂ reservoir for storage (below -130 ° C).

3. Thawing frozen PBMCs.

After setting the thermoblock at 37 ° C, the culture medium supplemented with 10% plasma was placed in a T flask. The volume of the culture medium can be adjusted according to the cell concentration, for example 4, 6, 8 or 10 ml. The cryogenic ampoule, frozen in example 2.2, was placed in a thermoblock for defrosting frozen PBMCs. After thawing, half of the frozen PBMCs were transferred to a T flask with culture medium. Then they were incubated in an incubator at 37 ° C with 5% CO ₂ during the day. The cultured PBMCs were collected in a tube, to which DPBS containing no Ca / Mg was added, and the tube was centrifuged for 5 min at 1500 rpm, and then the supernatant was discarded. The cells recovered by centrifugation were suspended in a small amount of culture medium, and the number of cells was counted. Cell viability after thawing is shown in table. 1. As you can see from the table. 1.93% or more PBMCs that were thawed from the frozen state of the cells survived, indicating high viability.

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Table 1. Viability of PBMC after thawing

Origin Viability(%) Donor 1 98.07 Donor 2 93 Donor 3 97.4

Example 2. Culturing NK cells.

2.1. Prepare a culture flask coated with fibronectin and gamma globulin.

2.1.1. Culture flask coated with fibronectin and gamma globulin (1).

0.1 ml of fibronectin (# FC-010, Millipore) and 0.121 ml of gamma globulin (# 020A1004, Greencross) were placed in a 15 ml tube and then 9.779 ml of Ca / Mg-free DPBS was added thereto. The resulting coating solution was pipetted into a T75 flask (# 156499, Nunc) and allowed to react at 2 to 8 ° C for 16 hours or longer. The remaining coating solution before cultivation was removed by washing with Ca / Mg-free DPBS.

2.1.2. Culture flask coated with fibronectin and gamma globulin (2).

A culture flask coated with fibronectin and gamma globulin was prepared in the same manner as in example 2.1.1, except that the amounts of fibronectin and gamma globulin in example 2.1.1 were only changed by 10 μl and 1.21 ml, respectively. ...

2.1.3. Fibronectin-coated culture flask.

0.2 ml of fibronectin (# FC-010, Millipore) was placed in a 15 ml tube and Ca / Mg-free DPBS was added thereto to bring the total volume to 10 ml. Then, a culture flask coated with fibronectin alone was prepared in the same manner as in Example 2.1.1.

2.2. First cultivation of NK cells.

2.2.1. First cultivation of NK cells (1).

The cell suspension obtained in example 1 was taken and placed in a coated flask prepared in example 2.1, which was supplemented with 1.5 ml of autologous plasma, 0.03 ml of an anti-NKp46 antibody solution (# MAB1850, R&D), 0.075 ml of IL- 18 (# B003-2, R&D) and 13.4625 ml Alys505NK-EX (# 01410P10, CSTI), then cultured in a CO ₂ incubator for 2-3 days. Thereafter, 1.5 ml of autologous plasma and 13.5 ml of Alys505NK-EX were added, then cultured in a CO2 incubator for a period of 1 day to 2 days.

2.2.2. First cultivation of NK cells (2).

All procedures were performed in the same manner as in example 2.2.1, except that 3 μl of an anti-NKp46 antibody solution (identical to above), 37.5 μl of IL-18 (identical to above) and Alys505NK-EX were added twice to a total volume of 35.95 ml.

2.2.3. First cultivation of NK cells (3).

All procedures were performed in the same manner as in example 2.2.1, except that 15 μl of an anti-NKp46 antibody solution (identical to the above), 7.5 μl of IL-12 (# 554613, BD) and Alys505NK-EX were added twice to a total volume of 26.98 ml.

2.2.4. First cultivation of NK cells (4).

All procedures were performed in the same manner as in example 2.2.1, except that 15 μl of an anti-NKp46 antibody solution (identical to above), 7.5 μl of IL-12 (identical to above), 37.5 μl were added IL-18 (identical to above) and Alys505NK-EX twice for a total volume of 26.94 ml.

2.2.5. First cultivation of NK cells (5).

All procedures were performed in the same way as in example 2.2.1, except that 0.03 ml of an anti-NKp46 antibody solution (identical to the above), 7.5 ml of IL-12 (identical to the above), 12, 5 μl IL-15 (# 247-1L-0025, R&D), 37.5 μl IL-18 and Alys505NK-EX twice for a total volume of 26.913 ml.

2.3. Second culturing of NK cells.

2.3.1. Second cultivation of NK cells (1).

After the first culturing according to example 2.2.1, the cultured cells in a T75 flask in an incubator were harvested and transferred to a T175 flask (# 159910, Nunc), which was supplemented with 3 ml of autologous plasma and 27 ml of Alys505NK-EX (# 01410P10, CSTI), cultured in CO ₂ incubator for a period of 1 day to 2 days. After that, 6 ml of autologous plasma, 0.12 ml of an anti-NKp46 antibody solution, 0.03 ml of IL-18 and 53.85 ml of Alys505NK-EX (# 01410P10, CSTI) were added, then cultured in a CO2 incubator for a period of 1 day up to 2 days.

2.3.2. Second cultivation of NK cells (2).

For the second cultivation of the culture obtained in example 2.2.2, all procedures were performed in the same manner as in example 2.3.1, except that 0.06 ml of an anti-NKp46 antibody solution (# MAB1850, R&D), 0, 06 ml IL-18 (# B003-2, R&D) and 53.88 ml Alys505NK-EX.

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2.3.3. Second cultivation of NK cells (3).

For the second cultivation of the culture obtained in example 2.2.2, all procedures were performed in the same manner as in example 2.3.1, except that 0.12 ml of an anti-NKp46 antibody solution (# MAB1850, R&D), 0, 03 ml IL-12 (# 554613, BD) and 53.85 ml Alys505NK-EX.

2.3.4. Second cultivation of NK cells (4).

For the second cultivation of the culture obtained in example 2.2.4, all procedures were performed in the same manner as in example 2.3.1, except that 0.06 ml of an anti-NKp46 antibody solution (# MAB1850, R&D), 0, 03 ml IL-12 (identical to above), 0.02 ml IL-18 (identical to above) and 44.89 ml Alys505NK-EX.

2.3.5. Second cultivation of NK cells (5).

For the second cultivation of the culture obtained in example 2.2.5, all procedures were performed in the same manner as in example 2.3.1, except that 0.06 ml of an anti-NKp46 antibody solution (# MAB1850, R&D), 0, 03 ml IL-12 (identical to above), 0.06 ml IL-15 (identical to above), 0.02 ml IL-18 (identical to above) and 62.83 ml Alys505NK-EX.

2.4. Third culturing of NK cells.

Cells from a T175 flask cultured in Example 2.3 and autologous plasma were added to the culture medium supplemented with 300 IU / ml IL-2 and cultured in a CO ₂ incubator. After 2-3 days, new culture medium (supplemented with 300 IU / ml IL-2) was mixed in equal volumes with the cell suspension in which the cells were cultured, and then cultured in a CO ₂ incubator.

In the aforementioned cultivation (in all cultivations - first, second and third), the IL-2-free culture medium for immune cells can be supplemented with a predetermined amount of IL-2 and then used instead of the culture medium supplemented with IL-2.

Example 3. Determination of proliferation and activation of cultured NK cells.

3.1. Determination of the total number of cultured cells and the order of increasing the number of NK cells.

All cells cultured in Example 2.4 were harvested and centrifuged for 5 min at 1500 rpm. Then the supernatant was discarded and the cells were suspended in phosphate buffered saline. 10 μl of the cell suspension was diluted with phosphate buffered saline and 10 μl of the diluted solution was mixed with 10 μl of trypan blue. The mixture was placed in a hemocytometer, the number of cells was counted and their viability was measured. The number of cells was calculated using the following formula: (number of living cells + number of dead cells) x1 / 4x2x dilution χ total volume x10 ⁴ , and cell viability was calculated using the following formula: number of living cells - (number of living cells + number of dead cells) x100.

The experimental conditions used in the present invention and the results are summarized in table. 2, fig. 2 and FIG. eight.

table 2

No. Flask coating mAb Cytokine Number of cells (x10 ⁷ cells) NK cells (%) Lysis K562 (%) Fibronectin Gamma globulin NKp46 IL-2 IL-12 IL-15 IL-18 The average SD The average SD The average SD one 0 0 68 9.8 21.3 5.1 0 0 2 O O O 96 11.12 25.6 3.4 0 0 3 O O O O 318 46.47 41.8 2.46 78 1.47 4 0 O 226 45.22 54.3 6.17 36 2.42 5 O O O 264 34.13 66.1 4.46 41 1.46 6 0 0 O 0 248 59.14 65.6 3.47 39 3.55 7 0 0 496 67.13 74.2 4.47 43 2.42 eight O 0 0 494 53.2 77.1 4.4 56 3.4 9 O O O 0 0 226 25.13 57.7 4.46 81.8 3.47 10 O O O 0 0 0 260 32.14 68.3 2.17 82.9 1.17 eleven O O O 0 0 0 0 250 44.14 68.9 3.14 76.9 2.17 12 O O O 0 0 504 45.14 78.1 3.47 82.7 1.46

Collectively, the combination of IL-2 and IL-18 was effective in increasing cell numbers and NK cell percentage. IL-12, IL-15, a mixture of IL-12 and IL-15, or IL-18 were effective in increasing the percentage of NK cells.

3.2. Determination of the effect of NK cell proliferation depending on the concentration of the first interleukin.

The cell suspension obtained in example 1 was taken and placed in the coated flask prepared in example 2.1. 1.5 ml of autologous plasma, 0.03 ml of an antibody solution to NKp46 (# MAB1850, R&D), 0.075 ml of IL-18 (# B003-2, R&D) and 13.4625 ml of Alys505NK-EX (# 01410P10, CSTI) without IL-2 and IL-2 was added at a concentration of 0 IU / ml to 10,000 IU / ml (Fig. 3), then cultured in a CO2 incubator for 14 days. After 14 days, the cultured cells were centrifuged for 5 min at 1500 rpm and the supernatant was discarded. The cells were suspended in phosphate buffered saline. 10 μl of the cell suspension was diluted with phosphate buffered saline and 10 μl of the diluted solution was mixed with 10 μl of trypan blue. The mixture was placed in a hemocytometer and the number of cells was counted. The number of cells was counted in the same way as in example 3.1. Number

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NK cells were counted based on the percentage of NK cells determined by flow cytometry. 100 IU / ml IL-2 was taken as 1 and the fold increase in the number of NK cells was calculated depending on the concentration of IL-2.

As shown in FIG. 3, at IL-2 concentrations ranging from 500 to 5000 IU / ml, a 6-fold or more increase in NK cell proliferation was observed compared to 100 IU / ml IL-2.

3.3 Determination of the activity of NK cells depending on the concentration of the second interleukin.

μl of fibronectin (# FC-010, Millipore) and 1.21 μl of gamma globulin (# 020A1004, Greencross) were placed in a 1.7 ml tube and then 97.79 μl of DPBS free of Ca / Mg was added thereto. The resulting coating solution was pipetted into a 96-well plate (# 167008, Nunc) and allowed to react at 2 to 8 ° C for 16 hours. The remaining coating solution was removed prior to cultivation by washing with Ca / Mg-free DPBS. ... The cell suspension obtained in example 1 was taken and added to a 96-well plate, and then 10 μl of autologous plasma, 0.2 μl of an anti-NKp46 antibody solution (# MAB1850, R&D), 89.9 μl of Alys505NK-EX (# 01410P10 , CSTI) with 1000 IU / ml IL-2 and IL-2 was added at a concentration of 0 to 10 ng / ml (see Fig.4A), and IL-18 was added at a concentration of 0 to 300 ng / ml (see Fig. Fig. 4B) and then cultured in a CO ₂ incubator for 48 hours. After 48 hours, the supernatant was transferred to a new tube and centrifuged for 5 minutes at 1500 rpm. Only the obtained supernatant was transferred to a new tube. The activity of NK cells was determined by the generated IFN-gamma using IFN-gamma in the EILSA kit (# DIF50, R&D).

As shown in FIG. 4A and B, high IFN-gamma production was observed at an IL-2 concentration of 0.1 to 10 ng / ml and an IL-18 concentration of 1 to 100 ng / ml.

3.4. Determination of the expression of the active receptor of cultured NK cells.

NK cell surface antigens collected in Example 2 were analyzed using flow cytometry. More specifically, the harvested cells were suspended in Ca / Mg-free DPBS and centrifuged for 5 min at 1500 rpm. Then the supernatant was discarded and the cell pellet was collected. Antibodies (CD3-FITC, CD56-APC, and CD16-PE) containing fluorescent materials were added to the pellet and incubated for 20 min at 4 ° C. FACS buffer was then added, centrifuged once for 1 min at 8000 rpm and analyzed by flow cytometry (FACSCalibur, BD). The results are shown in FIG. 5.

As shown in FIG. 5, cells proliferated according to a particular embodiment showed high levels of CD56 and CD16 expression and low CD3 expression compared to PBMCs prior to proliferation.

Expression of active NK cell receptors was investigated by flow cytometry. More specifically, the harvested cells were suspended in Ca / Mg-free DPBS and centrifuged for 5 min at 1500 rpm. The supernatant was then discarded and the cell pellet collected. Antibodies (KIRDL1-PE, NKG2A-PE, NKG2D-PE, NKp30-PE and NKp44-PE, NKp46-PE) containing fluorescent materials were added to the pellet and incubated for 20 min at 4 ° C. Then FACS buffer was added, the pellet was centrifuged once for 1 min at 8000 rpm and then analyzed by flow cytometry (Guava 8HT, MERK). The results are shown in FIG. 6.

As shown in FIG. 6, cells proliferated according to a particular embodiment showed a high level of expression of NKG2D, NKp30, NKp44, or NKp46 compared to PBMC before proliferation.

3.5. Analysis of the anticancer ability of cultured NK cells.

1) Analysis of the expression of anticancer substances.

The expression of anti-cancer substances by the NK cells obtained in example 2 was analyzed by flow cytometry. More specifically, the harvested cells were suspended in Ca / Mg-free DPBS and centrifuged for 5 min at 1500 rpm. The supernatant was then discarded and the cell pellet collected. The resulting cell pellet was treated with a fixing / permeabilizing solution (# 51-2090KZ, BD) and then treated with antibodies (Perforin-PE, GranzymeB-PE, and TRAIL-PE) containing fluorescent materials, respectively, and then incubated for 30 min at 4 ° C. Then there was added permeabilizing / washing buffer (# 51-2091KZ, BD) and centrifuged twice for 1 min at 8000 rpm, and then analyzed by flow cytometry (FACSCalibur, BD). The results are shown in FIG. 7.

As shown in FIG. 7, cells proliferated according to a particular embodiment showed a high level of expression of perforin, granzyme B or TRAIL compared to PBMC before proliferation.

2) Determination of the ability to kill cancer cells.

A K562 chronic myeloid leukemia cell line and various solid tumor cell lines (Hep3B, OVCAR3, HepG2, A704, and DU145) were harvested as target cancers and centrifuged for 5 min at 1500 rpm. All supernatants were discarded and each cell pellet was collected and washed with Ca / Mg-free DPBS. Culture medium was added to the washed cell pellet to prepare cells at a density of 5x105 cells / ml. The resulting cells were treated with 10 038848 CFSE (# c34554, Life technologies) and then incubated for 10 min in a CO ₂ incubator. The cells were washed twice with Ca / Mg-free DPBS and treated with prepared NK cells, and then incubated for 4 h in a CO ₂ incubator. Cells were treated with 7AAD 10 min before the end of incubation. After the end of the incubation, the cells were collected in a test tube. The ability of the resulting cells to kill cancer cells was then analyzed by flow cytometry (FACSCalibur, BD). The ability to kill cancer cells was calculated using the following equation.

Cytotoxicity (%) = (treated group of samples - spontaneous release) / (100 - spontaneous release) * 100

The resulting lysis of K562 is shown in FIG. 9 and in table. 2, and the results of lysis of various solid tumor cell lines are shown in FIG. 9.

Therefore, the combination of antibodies to NKp46 and flasks coated with fibronectin and gammaglobulin showed high cytotoxicity against K562, as well as various solid tumors, including OVCAR3 (ovarian cancer), Hep3V (liver cancer), HepG2 (liver cancer), A704 (kidney cancer ) and DU145 (prostate cancer) according to the E: T ratio (number of effector cells: number of target cells). The effector cells are NK cells and the target cells are cancer cells.

3.6. The result of culturing NK cells from frozen PBMCs.

PBMCs isolated from the blood of 3 donors and frozen PBMCs were cultured for 14 days in accordance with the proliferation method of the present invention, and their properties were analyzed using flow cytometry, examining the expression of CD3, CD16, CD19 and CD56 and cellular cytotoxicity to K562.

As a result, donor 1 showed a 968-fold increase in the number of NK cells, and the percentage of NK cells (CD3'CD56 ⁺ ) increased from 10.5 to 78.8% (Table 3, Figs. 10 and 11).

Table 3. The number and viability of cells cultured for 14 days from PBMC before cultivation and from frozen PBMC

No. Origin Target cell Total number of cells NK cells Viability (%) one Donor 1 PBMC 3x10 ' 3.2x10 ^b one hundred 14 days 3.9 ^x 10 ⁹ 3.1 x10 ⁹ 97.4 2 Donor 2 PBMC 3.33x10 ⁷ 5.1 x 10 ⁶ 93.69 14 days 5.4x10 ⁹ 4.2x10 ⁹ 96.73 3 Donor 3 PBMC 3x10 ⁷ 8.5x10 ⁶ 98.1 14 days 3.1 x10® 2.66x10 ⁹ 94.6

Donor 2 had an 823-fold increase in the number of NK cells (Table 3), and the percentage of NK cells (CD3'CD56 ⁺ ) increased from 15.2 to 77.2% (Table 4).

Table 4. Properties of cells cultured for 14 days from PBMC before cultivation and from frozen PBMC

No. Origin Cage-m ishen Determination of the phenotype (%) Cytotoxicity (%) CD3CD56 * CD16 ⁺ CD56 ⁺ CD3CD19 * one Donor 1 PBMC 10.5 11.2 8.08 4.03 14 days 78.8 86.1 0.34 82.36 2 Donor 2 PBMC 15.2 13.2 8.72 5.89 14 days 77.2 84.7 0.14 79.93 3 Donor 3 PBMC 28.4 5.88 16.6 6.8 14 days 85.8 90 0.07 90.6

The results indicate that NK cells can be obtained from frozen PBMCs by a method including freezing and thawing PBMCs and a method including culturing NK cells according to the present invention.

After reading the detailed description of the present invention, those skilled in the art will understand that the detailed description is only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the significant scope of the present invention is defined by the appended claims and their equivalents.

Claims (16)

CLAIM 1. A method for the proliferation of natural killer cells, the method comprising culturing natural killer cells in a culture medium containing IL-2 as the first interleukin - 11 038848 on; IL-18 as a second interleukin and anti-NKp46 antibody, wherein the cultivation is performed in the presence of gamma globulin and fibronectin. 2. The method of claim 1, wherein the proliferation of natural killer cells is the cultivation of peripheral blood mononuclear cells. 3. The method according to claim 2, wherein the peripheral blood mononuclear cells are in a thawed state after cryopreservation. 4. The method of claim 3, wherein the cryopreservation consists of a first freezing at a temperature of 4 to -42 ° C, a second freezing at a temperature of -42 to -15 ° C, and a third freezing at a temperature of -15 to -120 ° C. 5. The method according to claim 1, where the method of proliferation of natural killer cells includes the first cultivation of the cultivation of mononuclear cells of peripheral blood in a culture medium on a culture plate coated with gamma globulin and fibronectin; and a second cultivation, consisting in additionally introducing the culture medium into the culture obtained in the first culture, where the culture medium contains IL-2 as the first interleukin; IL-18 as a second interleukin; anti-NKp46 antibody and plasma. 6. The method of claim 5, wherein the plasma is isolated from peripheral blood from which peripheral blood mononuclear cells were obtained. 7. The method of claim 5, wherein the concentration of IL-2 in the culture medium is from 500 to 5000 IU / ml. 8. The method of claim 5, further comprising a third culturing culture by lowering the IL-2 concentration in the culture after the second culture to 4 / 5-1 / 10 or lower. 9. The method of claim 8, wherein the concentration of IL-2 in the culture medium in the third culture is between 100 and 1500 IU / ml. 10. The method of claim 1, wherein 70% or more of all natural killer cells proliferated according to the method express any one or more selected from the group consisting of CD56, CD16, NKG2D, perforin, and granzyme B. 11. A method for producing natural killer cells, the method comprising proliferating natural killer cells according to a method according to any one of claims 1-10 and preserving the proliferated cells after suspending in a solution containing dextran and albumin. 12. A pharmaceutical composition for the treatment or prevention of cancer, the pharmaceutical composition comprising natural killer cells proliferated according to a method according to any one of claims 1-11. 13. Composition for proliferation of natural killer cells, and the composition contains IL-2 as the first interleukin; IL-18 as a second interleukin; an anti-NKp46 antibody, wherein the composition further comprises gamma globulin and fibronectin. 14. The composition of claim 13, wherein the composition is for the proliferation of natural killer cells from peripheral blood mononuclear cells. 15. The composition of claim 14, wherein the peripheral blood mononuclear cells are thawed after cryopreservation. 16. The composition of claim 13, wherein the composition further comprises plasma isolated from peripheral blood from which peripheral blood mononuclear cells were obtained.