JP5358683B2 - Method of growing natural killer cells - Google Patents

Abstract

The present invention relates to an improved growth method for natural killer cells (NK cells). More specifically, the present invention relates to a growth method for natural killer cells, which comprises a step of culturing natural killer cells in a medium containing anti-CD3 antibodies and interleukin proteins in the presence of peripheral blood leukocytes. The present invention provides an innovative growth method for natural killer cells, which can obtain a large amount of natural killer cells by remarkably increasing the growth rate compared with conventional growth methods for natural killer cells.

Description

  The present invention relates to a method for growing natural killer cells (NK cells) with high yield, and more specifically, a medium containing anti-CD3 antibody and interleukin protein in natural killer cells. And a method for growing natural killer cells, comprising a step of culturing in the presence of peripheral blood white blood cells.

  Natural killer cells (hereinafter sometimes abbreviated as “NK cells”) are lymphocyte cells that play a role in immune responses. Although this cell has various functions, it has a strong activity to kill tumor cells in particular, so it is considered to be an important member of the immune surveillance mechanism that removes cells that are tumoried or have abnormalities toward tumorigenesis in the body. ing. As a result, researches have been conducted for a long time to effectively use these cells in the treatment of tumors and the removal of virus-infected cells that are supposed to be the source of tumors.

  Most NK cells present in a healthy person are in an inactivated state. Many researchers are working on activating NK cells from normal blood or from the blood of inactivated patients.

  The high cytotoxicity of NK cells activated in vitro opens up the potential as an immunocytotherapeutic agent, confirming the potential for various carcinomas in vitro or in animal experiments. Usually, tumor cell lines are used to confirm the cytotoxicity of NK cells in vitro, and against various cancer types such as blood cancer, liver cancer, lung cancer, kidney cancer, childhood neuronal cancer and skin cancer. NK cells showed considerable cytotoxicity. In particular, NK cells showed positive therapeutic effects in both clinical and nonclinical settings for hematological cancer and childhood neuronal carcinoma.

  Despite these clinical possibilities, the number of NK cells present in the body is not so large, and the number of effective NK cells necessary to show a therapeutic effect is very large. ) To collect a large amount of white blood cells, only one or two treatments are possible with a single blood collection.

  Sufficient number of cells and repeated administration are very important factors in cell therapy therapy, but NK cell proliferation is incomplete and a sufficient number of cells are not administered. There have been many studies on the proliferation of NK cells to overcome these problems, but they have not reached clinically applicable levels. In general, studies on the proliferation of NK cells using IL-2 or other cytokines and chemicals have only increased the number of initially isolated NK cells by about 3 to 10 times.

  Since the 1990s, research on the growth of natural killer cells has been promoted in various directions. IL-15 (Dunne J et al., Immunology, vol. 167: 3129. 2001, SA Perez, et al., Blood, vol) as well as IL-2 used for the proliferation / activity of existing T cells 106: 158, 2005), LPS (MR Goodier et al., Immunology 165: 139,2000), and OKT-3 antibody (Condiotti R, et al., Experimental Hematology 29: 104, 2001) that stimulates CD3. There has been research to grow natural killer cells by using them in single / complex form. However, these are variations on and evolution of the classic use of IL-2 and have only found new growth materials and have not been able to present innovative growth methods.

  On the other hand, there have been reports of cases where NK cells were amplified using tumor cell lines as feeder cells by some researchers. It was an unsuitable way to ensure important safety in application.

  Therefore, the present inventors ensured safety by using inactivated autologous peripheral blood cells as support cells, and by simultaneously treating OKT-3 antibody and IL-2, the killing ability of natural killer cells is While maintaining this, it was confirmed that the proliferation rate increased steadily, and the present invention was completed.

  An object of the present invention is to provide a method for growing natural killer cells with high efficiency.

  In order to achieve the above object, the present invention comprises a step of culturing natural killer cells in a medium containing anti-CD3 antibody and interleukin protein in the presence of peripheral blood leukocytes (PBL). A method for explosively growing high purity natural killer cells in a short period of time is provided.

  As a preferred example, in the natural killer cell culture, the OKT-3 antibody and IL-2 are simultaneously treated while using autologous PBL (peripheral blood lymphocyte) as a feeder cell.

Further, the method includes a step of removing anti-CD3 antibody from the culture and a step of further culturing by adding the culture solution from which the anti-CD3 antibody has been removed to a medium containing interleukin protein. However, at this time, the culture solution from which the anti-CD3 antibody added for additional culture has been removed preferably contains natural killer cells at a concentration of 1 × 10 ^{5 to} 3 × 10 ⁶ cells / well. .

It is the result of having analyzed the surface type of the initial NK cell separated from the peripheral blood of a healthy person. It is the result of having analyzed the surface type of the NK cell obtained by culture | cultivating by the method of this invention. It is the result of having confirmed the tumor killing ability of the NK cell obtained by culture | cultivating by the method of this invention. It is the result of having confirmed the proliferation ability of the NK cell obtained by culture | cultivating by the method of this invention. It is the result of having compared the proliferative ability of the NK-cell obtained by culture | cultivating on each culture condition by the presence or absence of supporting cell PBMC, anti-CD3 antibody OKT-3, and IL-2. It is the result of having compared the proliferative ability of NK-cells obtained by culturing while changing the seeding concentration of natural killer cells.

  The present invention will be specifically described below.

  From one aspect, the present invention cultivates natural killer cells (NK) in a medium containing anti-CD3 antibody and interleukin protein in the presence of peripheral blood white blood cells (PBL). The present invention relates to a method for growing natural killer cells.

  The method for growing the natural killer cells of the present invention is not particularly limited, and can be performed, for example, including the following steps.

(1) a step of separating peripheral blood white blood cells and natural killer cells from human peripheral blood,
(2) a step of inactivating autologous peripheral blood white blood cells from which natural killer cells have not been separated (a step of preparing inactivated support cells),
(3) a step of culturing natural killer cells in the presence of peripheral blood leukocytes inactivated in a medium containing anti-CD3 antibody and interleukin protein,
(4) removing anti-CD3 antibody from the culture, and
(5) A step of performing additional culture by adding the culture solution from which the anti-CD3 antibody has been removed to a medium containing interleukin protein.

  Natural killer cells are present in about 10-15% in the blood of healthy individuals and have a high killing ability when reacting with non-self antigens. In the penetration of cells and bacteria infected by various viruses, or the generation of abnormal cells, NK cells react non-specifically and remove foreign substances. However, the number of NK cells present in the body is not so large, and the number of effective NK cells required to show a therapeutic effect needs to be considerably large, so there is a need for an effective NK cell proliferation method. The reality is that sex is required.

  There are two main methods for growing natural killer cells. After purely isolating NK cells, a method of amplifying them by applying appropriate stimuli while using supporting cells, and from whole peripheral blood mononuclear cells (PBMC) or peripheral blood mononuclear cells (PBMC) There is a method of selectively amplifying NK cells to obtain relatively many NK cells.

  Cells obtained by selective amplification of natural killer cells from PBL without isolation of natural killer cells are inferior in cell killing ability to the isolated natural killer cells, and only natural killer cells However, since T cells also exist, there is a problem that it is limited to self-transplantation unless T cells that recognize self and non-self are removed by self MHC molecules.

  The present invention relates to the former method for amplifying isolated natural killer cells, and is characterized in that support cells are also used in the proliferation method according to the present invention.

  As a method for separating natural killer cells from peripheral blood, a normal method known to those skilled in the art can be used, and a commercially available one can also be purchased and used. In one embodiment of the present invention, Rosettesep NK cell enrichment cocktail (Stem cell technologies, 15065) was purchased and used.

  On the other hand, "feeder cells" are cells that cannot divide and proliferate, but because they have metabolic activity, they produce various metabolites that help the target cells grow. “Supporting cells”. In the present invention, the term “feeder cell” is used as the term supporting cell.

  The feeder cells used in the present invention include animal cell lines into which genes have been introduced and peripheral blood leukocytes treated with various cytokines or compounds (PBL), self or other peripheral blood leukocytes (PBL), Examples thereof include T-cells, B-cells, and mononuclear cells. Most preferably, autologous peripheral blood white blood cells (PBL) can be used.

  Autologous peripheral blood white blood cells used for the supporting cells are inactivated to ensure safety. As a method for inactivation, a normal method known to those skilled in the art may be used. For example, a method of irradiating γ rays can be used. Such inactivated feeder cells include purified T-cells.

  As in the present invention, the growth method using feeder cells is a method in which natural killer cells are purely isolated and then proliferated, and has an advantage that only the natural killer cells that have been isolated can be proliferated.

  Furthermore, the growth method of the present invention is characterized in that natural killer cells are cultured in a medium containing an anti-CD3 antibody and an interleukin protein.

  An anti-CD3 antibody is a protein that specifically reacts with the CD3 antigen, which is a group of molecules that form an antigen recognition complex by associating with the T cell receptor (TCR). This region is long and plays a role in transmitting an antigen recognition signal into cells.

  Examples of anti-CD3 antibodies that can be used in the present invention include OKT-3 antibody, UCHT1 antibody, HIT3a antibody, and the like, preferably OKT-3 antibody.

  Interleukin (IL) protein is a general term for proteinaceous biologically active substances produced by immunocompetent cells such as lymphocytes or monocytes and macrophages, and refers to a group of molecular species in cytokines.

  Examples of interleukin proteins that can be used in the present invention include IL-2, IL-15, IL-12, IL-18, and IL-21, and IL-2 protein is preferred.

  In the culture method of the present invention, NK cells and PBL isolated from human peripheral blood are added to a normal animal cell culture medium such as AIM-V medium, RIMI1640, CellGro SCGM, and X-VIVO20, and the culture is treated with -Add CD3 antibody and interleukin protein and culture. In one specific example of the present invention, OKT-3 antibody and IL-2 were added and cultured. The concentration of the added OKT-3 antibody is 0.1 to 100 ng / ml, preferably about 10 ng / ml, and the concentration of IL-2 is 10 to 2000 U / ml, preferably about 500 U / ml.

  Moreover, it can also culture | cultivate by adding the additional growth factor which supports proliferation of serum or plasma and a lymphocyte here. The kind of serum or plasma to be added to the medium is not particularly limited, and commercially available animal-derived ones can be used. For example, a method known to those skilled in the art can be used, such as adding a combination of cytokines for proliferating lymphocytes from PBMC or lectins that stimulate lymphocyte proliferation.

  From another point of view, the present invention provides an optimal culture concentration of natural killer cells that can proliferate natural killer cells significantly.

  As described above, one embodiment of the present invention will be described in more detail. First, isolated natural killer cells are cultured in a medium containing anti-CD3 antibody and interleukin protein in the presence of peripheral blood white blood cells. Culturing in step (b), removing the anti-CD3 antibody from the culture, and adding the culture solution from which the anti-CD3 antibody has been removed to a medium containing an interleukin protein for additional culture.

  At this time, when the culture solution from which the anti-CD3 antibody has been removed is added to the medium containing the interleukin protein and further cultured, the concentration of the natural killer cells seeded on the medium greatly affects the growth rate.

Preferably, natural killer cells are seeded at a concentration of 1 × 10 ^{5 to} 1 × 10 ⁶ cells / well, more preferably a concentration of 1 × 10 ^{5 to} 3 × 10 ⁶ cells / well. In particular, when seeded at a concentration of 2 × 10 ⁵ cells / well, it was confirmed through experiments that the growth rate was about 900 times 14 days after culturing.

  In the present invention, natural killer cells are treated with an anti-CD3 antibody such as OKT-3 antibody and an interleukin protein such as IL-2 at the appropriate concentration while using supporting cells, so that only supporting cells are treated. Compared to existing studies using only or stimulation of OKT-3 antibody, high purity natural killer cells can be proliferated more explosively in a short period of time.

  From another viewpoint, the present invention relates to a natural killer cell obtained by the above method. The surface type characteristics of natural killer cells grown and cultured by the above method will be described below.

  More than 90% of the primary NK cells from which the peripheral blood of healthy individuals is isolated have a surface type of CD3- / CD56 +. When this is grown and cultured by the method of the present invention, the number of CD3 + T cells is relatively decreased around the seventh day from the culture start date, the number of CD3- / CD56 + NK cells is further increased, and from the culture start date. Around day 10, almost all CD3 + T cells disappear, and more than 95% of all cells are activated NK cells that express CD16. That is, high-purity natural killer cells having a CD16 + surface type can be obtained by growing in a short period of time.

  Therefore, it is possible to prepare a cell therapeutic agent effective for removing a virus-infected cell that is supposed to be a source of tumor treatment or tumor using a large amount of activated NK cells that can be clinically applied.

  Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are merely illustrative of the invention and that the scope of the invention is not to be construed as limited by these examples.

Example 1: Preparation of feeder cells and separation of natural killer cells
(1) Preparation of supporting cells 20 ml of peripheral blood of healthy subjects was collected, and 5 ml of the collected blood was placed in a 15 ml conical tube. An additional 5 ml of saline was added to the blood and mixed well with a pipette. Place 5 ml of ficoll (GE Healthcare, Uppsala, 17-1440-03) into a new 15 ml conical tube and mix (diluted) as above into a 15 ml tube containing ficoll. After blood was carefully added, it was centrifuged at 2000 rpm for 30 minutes at room temperature (Hanil, Korea, Union 32-R).

  The immune cell layer formed between Ficoll and plasma was transferred to a new 15 ml conical tube, HBSS was added to a total volume of 10 ml, the cells were mixed well, and centrifuged at 1200 rpm for 10 minutes. The supernatant was sufficiently removed by vacuum suction. Furthermore, the process of adding 10 ml of HBSS and centrifuging was repeated.

Add 1 ml of AIM-V media (Invitrogen, 12055091) containing 5% of cell culture medium (hAB serum (Sigma, H4522)) Transfer to a tube, add 90 μl trypan blue (Gibco), mix well with pipette, stain with trypan blue (Gibco, 15250-061) reagent and inverted microscope (Olympus, CK2 -TRC-2) was used to dilute the cell culture solution so that the number of cells was 5 × 10 ⁶ cells / ml.

  Transfer 1 ml cell suspension to a 5 ml tube for FACS analysis and inactivate the remaining cells with 2000 cGy gamma radiation (gamma-irradiatrior, MDS Nordion, gammacell 3000 Elan) Prepared.

(2) Isolation of NK cells Transfer 15 ml of blood collected from healthy subjects before to a new 50 ml conical tube, add Rosettesep NK cell enrichment cocktail (Stem Cell, 15065), and slowly add 20 minutes at room temperature. The reaction was carried out while rotating. To the blood after the above reaction was completed, 15 ml of physiological saline was added and mixed well.

  Place 5 ml of ficoll into each of 3 new 15 ml conical tubes, carefully add 10 ml of blood mixed with the above physiological saline into the 15 ml conical tube containing ficoll, and then add 30 ml at room temperature at 2000 rpm. Centrifuged for minutes. After centrifugation, all natural killer cell layers between Ficoll and autologous plasma were transferred to a fresh 15 ml conical tube, HBSS was added to a total volume of 10 ml, and further centrifuged at 1500 rpm for 10 minutes. The supernatant was sufficiently removed, and HBSS was further added to a total volume of 10 ml. The cells were sufficiently separated, and then centrifuged at 1200 rpm for 10 minutes. After removing the supernatant under vacuum, 1 ml of cell culture was added to sufficiently separate the cells.

Transfer 10 μl of the cell dilution to a microtube, add 40 μl of trypan blue, mix well with a pipette, stain with trypan blue, and measure the number of cells while culturing so that the number of cells becomes 1 × 10 ⁶ cells / ml. Diluted with liquid.

(3) Characteristics of isolated early NK cells The isolated NK cells were stained with anti-human CD3-FITC and anti-human CD56-APC antibodies to analyze the surface type. As a result, as can be seen in FIG. 1, it was confirmed that 90% or more of the initially isolated cells were CD3- / CD56 + NK cells.

Example 2: Culture of isolated natural killer cells Initial cells were cultured in 12-well plates (Falcon). 500 μl of the feeder cells prepared in Example 1- (1) was placed in a well, and 500 μl of the natural killer cells separated in Example 1- (2) were additionally placed in the well containing the feeder cells.

  Carefully place the plate in each well containing cells with 500 ng / ml IL-2 (Norvatis cytokine and OKT-3 antibody (ebioscience, 16-0037) 10 ng / ml. Shake to mix cells and cytokines well.

  The plate was placed in a 37 ° C. humidified incubator containing 5% carbon dioxide and cultured for 5 days. At this time, no culture medium or cytokine was added.

  On the fifth day from the culture start date, all wells containing cells were pipetted and the cells were collected in a 15 ml conical tube. 1 ml each of cell culture solution was added to each well from which cells had been removed, and the remaining cells were sufficiently recovered, and the recovered cells were centrifuged at 1200 rpm for 10 minutes. The supernatant was sufficiently sucked in vacuum to remove the OKT-3 antibody.

The remaining cells were diluted by adding 2 ml of cell culture solution, and 10 μl of the diluted cells was taken into a microtube, mixed well with 90 μl of trypan blue solution, stained with trypan blue, and the number of cells was measured. Cell culture medium was added and diluted to 2 × 10 ⁵ cells / well.

Further, IL-2 was added to 500 U / ml and mixed well with the cells, and then cells were seeded in a 12-well plate at 2 × 10 ⁵ cells / ml / well. The plate was placed in a 37 ° C. wet incubator containing 5% carbon dioxide and further cultured for 12 days.

  At this time, 1 ml of a cell culture solution supplemented with 500 U / ml IL-2 was added from the day after the removal of the OKT-3 antibody to the 10th day from the start of the culture.

On the 10th day after the start of the culture, the cells are collected, the number is measured, and the collected cells are re-planted with 1 × 10 ⁷ cells in a T75 flask and 500 U / ml IL-2. 5 ml of cell culture solution containing was added. 5 ml of cell culture medium was added every day until it was transferred to the next flask.

  After no more cells were grown, the cells were transferred to a new flask, and 5 ml of cell culture medium containing IL-2 (500 U / ml) was added daily until the 17th day.

Example 3: Surface type analysis of the obtained NK cells
After removal of OKT-3, IL-2 alone was treated alone, and on the 7th and 10th days, some cells were collected and analyzed for surface type.

Before culturing, during or after culturing, the cells were collected and centrifuged at 12000 rpm for 5 minutes, and the culture solution was removed by vacuum suction. The number of cells was measured by diluting with 1 ml of FACS buffer (2.5% FBS + PBS), and diluted with FACS buffer to 5 × 10 ⁶ cells / ml. 100 μl each of the diluted cell solution was placed in a FACS tube (Falcon), and the antibody was placed as follows.

Tube 1: No staining Tube 2: Anti-human CD3-FITC (BD Pharmingen, 5555339) + Anti-human CD56-APC (BD Pharmingen, 555518) + Anti-human CD16-PE (BD Pharmingen, 555407)
Tube 3: Anti-CD16-FITC (Color control) (BD Pharmingen, 555406)
Tube 4: Anti-CD56-PE (Color control) (BD Pharmingen, 555516)
Tube 5: Anti-CD56-APC (Color control)
The tube was allowed to stand at refrigeration for 30 minutes for staining, and then 2 ml of FACS buffer was added to the stained cells and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, 2 ml of buffer was added, and the mixture was centrifuged at 1500 rpm for 5 minutes. Further, the supernatant was removed, and 300 μl FACS buffer was added, and cells were separated by vortexing. Surface type was analyzed using FACSCalibur (Becton Dickinson).

  As a result, as shown in FIG. 2, those treated with OKT-3 on day 7 had relatively fewer CD3 + T cells than those cultured without treatment, and more CD3-CD56 + NK cells. There were many. CD3 + cells are presumed to be irradiated PBMC that have not yet died. When cultivated with OKT-3 and feeder PBMC on the 10th day from the start of culture, almost all CD3 + T cells disappeared and 95% or more of the cells were confirmed to be NK cells. The That is, all the expanded NK cells are activated NK cells that express CD16.

Example 4: Evaluation of cell killing ability of cultured NK cells (Cr-release assay)
(1) Preparation of effector cells Some cells were collected on the 14th day of natural killer cell culture, centrifuged at 1200 rpm for 5 minutes to remove the supernatant, and then 2 ml of cell culture solution. And diluted. Cell culture medium was added and diluted so that the number of cells was 3 × 10 ⁶ cells / ml, and then adjusted to use a working cell: target cell (E: T) ratio = 30: 1. .

  Take 1 ml of the cell suspension prepared as described above, put it in a new tube, add 2 ml of cell culture medium and mix well, then work cell: target cell (E: T) ratio = 10: Combined to use for 1. Also, take 500 μl of the cell suspension, put it in a new tube, add 4.5 ml of cell culture medium and mix well, then use it at a working cell: target cell (E: T) ratio of 3: 1. It was adjusted.

  100 μl of NK cells adjusted at the specific ratio described above were added to a 96-well plate at 3 wells / rate for each condition.

(2) Target cell preparation
Prepare two cells by preparing CEM, which is an acute lymphoblastic leukemia cell line that is 80% confluent, and K562, a cell line that is chronic myelogenous leukemia (CML). After recovering the strain, it was placed in a 15 ml conical tube and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, and 5 ml of cell culture medium was added to dilute the cells. The number of cells was measured and 1 × 10 ⁶ cells were transferred to a 15 ml tube. The cell culture solution was added to the transferred cells so that the total volume became 10 ml, and then centrifuged at 1200 rpm for 5 minutes. The supernatant was removed and 25 μl of FBS was added to dilute the cells, and then 100 μl of Cr-51 (Perkin Elmer) was added.

  The tube was placed in a 37 ° C. humidified incubator containing 5% carbon dioxide and allowed to stand for 1 hour, and then the cells were taken out and the cell culture solution was added to a total volume of 10 ml and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed and washed twice more in the same manner. 10 ml of the cell culture solution was added to the washed cells, and diluted uniformly with a pipette.

(3) Measurement of killing ability The diluted cell line was further added in an amount of 100 μl per well into a 96-well plate (Falcon) having a round bottom containing effector cells prepared in advance. As a negative control for each condition, 100 μl of target cells were placed in three wells without working cells, and 100 μl of cell culture medium was placed. As a positive control for each condition, put 100 μl of target cells in 3 wells without working cells, add 100 μl of PBS containing 1% triton X-100, Cultured for 4 hours.

  Thereafter, the cells were precipitated by centrifugation at 2000 rpm for 3 minutes, 100 μl of the supernatant was transferred to a 5 ml test tube, and gamma rays were measured using a gamma-counter (COBRA). Cytotoxicity was calculated using the following formula:

  As a result, as can be seen from FIG. 3, the cytotoxicity between the two cell lines was different, but all of the two types of target cells showed high cytotoxicity of up to 70% or more.

Example 5: Evaluation of cell proliferation ability of cultured NK cells (CFSE-proliferation assay)
As in Example 1, NK cells were separated from 15 ml of peripheral blood of healthy subjects using RosetteSep, and autologous peripheral blood cells were irradiated to suppress proliferation and used as support cells. Anti-CD3 antibody (OKT-3) was stimulated at a low concentration for 5 days for PBL stimulation, and then cultured for 17 days in a medium supplemented with IL-2 to confirm up to 600 times the proliferation of NK cells.

  As a result, as can be confirmed from FIG. 4, when there is no stimulation with OKT-3 antibody, it ends with 20-fold growth after 10 days of culture, whereas 112-fold after 10 days of culture with OKT-3 stimulation, After 17 days of culture, up to 600-fold growth was confirmed.

Comparative Example 1: Comparison of cell proliferation ability of cultured NK cells
In order to compare and evaluate the proliferative ability of NK cells, the cells were cultured under the following conditions.

a. NK cells + IL-2 (500 U / ml)
b. NK cells + IL-2 (500 U / ml) + OKT-3 (10 ng / ml)
c. NK cells + IL-2 (500 U / ml) + irradiated PBMC
d. NK cells + IL-2 (500 U / ml) + irradiated PBMC + OKT-3 (10 ng / ml)
All NK cells were isolated using Rosettesep as in Example 1, and irradiated PBMCs were seeded at 5 times the number of NK cells. On the fifth day from the culture start date, each cell was collected and the number of cells was measured.

Prepare 1 × 10 ⁶ cells according to the above conditions, transfer them to a 5 ml tube, put the cell culture solution to a final volume of 500 μl, add 5 μM CFSE solution, and then add 30 Left for a minute. After washing 3 times with PBS, the wavelength of 530 nm was analyzed using FACS Calibure.

  As a result, as can be seen from FIG. 5, culturing with supporting cells promotes cell proliferation more than culturing only NK cells alone, and treatment with OKT-3 together with supporting cells is more effective for cell proliferation. I confirmed that there was. The most prominent effect is achieved when IL-2 and OKT-3, which are the above-mentioned conditions d, are treated at the same time, about 200 times in the culture for 14 days, and about 600 times in the culture for 17 days. The above proliferation ability was shown.

Example 7: Evaluation of cell proliferation ability according to concentration of cultured NK cells After performing initial culture and removal of OKT-3 as in Example 2, the number of NK-cells varies according to different concentrations as follows: After the cells were diluted, they were seeded in a 12-well plate, and further cultured for 9 days in a cell incubator to measure the number of cells.

a. 1 × 10 ⁵ cells / ml / well
b. 2 × 10 ⁵ cells / ml / well
c. 5 × 10 ⁵ cells / ml / well
d. 1 × 10 ⁶ cells / ml / well As a result of comparing cell growth under each condition, as shown in FIG. 6, when seeded at 2 × 10 ⁵ cells / ml, the maximum growth was about 900 times in 14 days. When seeded at 1 × 10 ⁶ cells / ml, the degree of proliferation was the lowest at about 100 times in 14 days. Therefore, after removing OKT-3, it was confirmed that the seeding concentration acts as a very large factor in the growth of natural killer cells.

  As described above, the details of the present invention have been described in detail, and it is obvious for those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

  The natural killer cell growth method according to the present invention is an epoch-making method capable of maximizing proliferation while maintaining the killing ability of natural killer cells as compared with the existing methods, so that many natural killer cells can be obtained with a small amount of blood. By being able to obtain cells, it is useful for regular use as a cell therapeutic agent.

  As described above, specific portions of the present invention have been described in detail. For those skilled in the art, such specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereby. it is obvious. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (7)

  A step of culturing natural killer cells in a medium containing an anti-CD3 antibody for stimulating the supporting cells and an interleukin protein as a growth factor in the presence of peripheral blood leukocytes inactivated as supporting cells. A method for growing natural killer cells, comprising:   2. The method for growing natural killer cells according to claim 1, wherein the anti-CD3 antibody is selected from the group consisting of OKT3, UCHT1 and HIT3a.   3. The method for growing natural killer cells according to claim 2, wherein the anti-CD3 antibody is an OKT-3 antibody.   2. The method for growing natural killer cells according to claim 1, wherein the interleukin protein is selected from the group consisting of IL-2, IL-15, IL-12, IL-18 and IL-21.   5. The method for growing natural killer cells according to claim 4, wherein the interleukin protein is interleukin-2 (IL-2).   2. The method for growing natural killer cells according to claim 1, wherein the inactivated peripheral blood white blood cells include T cells (purified T cells). 2. The method of proliferating natural killer cells according to claim 1, wherein the peripheral blood white blood cells are autologous peripheral blood white blood cells collected from the same individual from which the natural killer cells were collected .