KR20100012586A - Method for proliferating natural killer cell - Google Patents

Abstract

PURPOSE: A method for proliferating natural killer cells(NK cells) is provided to enhance proliferation rate of the NK cells and commercialize as a therapeutic agent. CONSTITUTION: A method for proliferating natural killer cells(NK cells) comprises: a step of culturing NK cells in a medium containing anti-CD3 antibody and interleukin protein under the presence of peripheral blood leukocytes; a step of removing anti-CD3 antibody from the culture medium; and a step of adding culture liquid in which anti-CD3 antibody is removed and culturing the cells.

Description

Method for Proliferating Natural Killer Cells {Method for Proliferating Natural Killer cell}

The present invention relates to a method for propagating natural killer cells (NK cells) in high yield, and more specifically, natural killer cells are peripheral blood leukocyte cells in a medium containing anti-CD3 antibody and interleukin protein. It relates to a method for propagating natural killer cells, including the step of culturing in the presence of.

Natural killer cells (hereinafter sometimes abbreviated as "NK cells") are lymphocytic cells that play a role in the immune response. Although these cells have many functions, in particular, they have a strong activity of killing tumor cells, and thus are thought to be important members of the immune monitoring apparatus for removing cells with tumorous or tumorigenic abnormalities in the body. Therefore, research has been conducted for a long time to effectively use these cells for tumor treatment and removal of virus-infected cells that are supposed to be the source of tumors.

Most of the NK cells in the normal body are in an inactive state. Many researchers have been working to activate NK cells from normal or inactivated patient blood.

The high cytotoxicity of NK cells activated in vitro opens up the possibility of immunocytotherapy and has been confirmed in vitro or in animal experiments on various carcinomas. Normally, tumor cell lines were used to identify in-vitro cytotoxicity and showed significant cytotoxicity against various cancers including hematologic cancer, liver cancer, lung cancer, kidney cancer, pediatric nerve cancer and skin cancer. In particular, hemologic and pediatric neurocarcinomas showed clinical and non-clinical effects.

Despite these clinical possibilities, the number of NK cells present in the body is not very high, and the number of effective NK cells required for the therapeutic effect is very high. Therefore, leukapheresis is performed to collect a large amount of leukocytes. Even if a single blood collection can only be one or two treatments.

Sufficient cell dose and repeat dosing are very important factors in cell therapy. However, when NK cells do not proliferate due to insufficient growth of NK cells, the biggest problem applied to clinical practice is In order to overcome this problem, many studies on NK cell proliferation have been made, but they have not reached the level applicable to the clinical practice. In general, the study of NK cell proliferation using IL-2 or other cytokines and chemicals has not reached an increase of 3 to 10 times the number of initially isolated NK cells.

Since the 1990s, research on the proliferation of natural killer cells has been conducted in various fields. IL-15 (Dunne J et al., Immunology, vol. 167: 3129. 2001; SA Perez, et al., Blood, vol. 106: 158, 2005) as well as IL-2, which has been used for conventional T cell proliferation / activity ), LPS (MR Goodier et al., Immunology 165: 139, 2000), CDT-stimulating OKT-3 antibody (Condiotti R, et al., Experimental Hematology 29: 104, 2001) in single / complex form There has been research to propagate natural killer cells by use. However, they only found new proliferative substances in the form of modifications and developments to the classical use of IL-2, but did not suggest breakthrough proliferation methods.

On the other hand, some researchers have reported cases of amplifying NK cells using tumor cell lines as feeder cells, but most of them are suitable for ensuring important safety for clinical applications, such as using tumor cell lines as support cells. It did not use the method.

Therefore, the present inventors secured safety by using inactivated autologous peripheral blood cells as feeder cells, and simultaneously treated with OKT-3 antibody and IL-2 to maintain natural killer cell killing ability while proliferating rate significantly. The increase was confirmed, and the present invention was completed.

It is an object of the present invention to provide a method for propagating 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 the presence of peripheral blood lymphocytes (PBL) in a medium containing anti-CD3 antibody and interleukin protein, high purity natural Provided is a method for explosively proliferating killer cells.

In a preferred embodiment, the present invention simultaneously treats OKT-3 antibody and IL-2 while using natural PBL (peripheral blood lymphocyte) as a feeder cell in culture of natural killer cells.

In addition, the method includes removing the anti-CD3 antibody from the culture and adding the culture medium from which the anti-CD3 antibody is removed to the medium containing the interleukin protein, wherein further culturing is performed. It is preferable that the culture medium from which the anti-CD3 antibody to be added is removed contains natural killer cells at a concentration of 1 x 10 ⁵ to 3 x 10 ⁶ cells / well.

Natural killer cell proliferation method according to the present invention is a breakthrough method capable of maximal proliferation while maintaining the killer ability of natural killer cells, compared to the conventional method, it is possible to obtain a large number of natural killer cells with a small amount of blood collection It is useful for commercialization as a therapeutic agent.

In one aspect, the present invention includes a step of culturing a natural killer cell (NK cell) in the presence of peripheral blood lymphocytes (PBL) in a medium containing anti-CD3 antibody and interleukin protein. It relates to a method for propagating natural killer cells.

The natural killer cell proliferation method of the present invention is not particularly limited and may be performed, for example, including the following steps:

(i) separating the peripheral blood leukocyte cells and natural killer cells from human peripheral blood;

(ii) inactivating autologous peripheral white blood cells that did not separate natural killer cells (preparing inactivated support cells);

(iii) culturing the natural killer cells in the presence of inactivated peripheral blood leukocyte cells in a medium containing anti-CD3 antibody and interleukin protein;

(iv) removing the anti-CD3 antibody from the culture; And

(v) adding the culture medium from which the anti-CD3 antibody is removed to the medium containing the interleukin protein to further culture.

Natural killer cells (NK cells) are present in about 10-15% of normal blood and have high killing capacity when reacted with non-self. In cells infected with various viruses, invading bacteria, or generating abnormal cells, NK cells react nonspecifically and immediately remove foreign substances. However, since the number of NK cells present in the body is not very high, and the number of effective NK cells required for showing a therapeutic effect should be very large, there is a need for an effective NK cell proliferation method.

There are two ways to multiply natural killer cells. There is a method of purely separating NK cells and then amplifying them with appropriate stimulation by using a feeder cell. The peripheral blood mononuclear cells (PBMC) or peripheral blood mononuclear cells (PBMC) cell) is a method of obtaining a relatively large number of NK cells by selectively amplifying NK cells.

Cells obtained by selectively amplifying spontaneous killer cells from PBL without separation of spontaneous killer cells have lower cell killing capacity than pure spontaneous killer cell populations, and autologous MHC is present because not only pure killer cells but also T cells exist. As long as the molecules do not remove T cells that recognize self and non-self, there is a problem that only the transplantation can be limited.

The present invention relates to a method for amplifying an isolated natural killer cell (NK cell) of the former, characterized in that the proliferation method according to the present invention also uses a feeder cell.

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

On the other hand, "Feeder cell" is a cell that prevents division and proliferation but has metabolic activity and produces various metabolites to help the growth of the target cell. do. In the present invention, the term 'feeder cell' is also used as a term support cell.

As feeder cells used in the present invention, animal cell lines into which genes have been introduced, peripheral blood leukocyte cells (PBL) treated with various cytokines or compounds, or peripheral blood leukocyte cells (PBLs) of self or others ), T-cell, B-cell, or monocytes. Most preferably, magnetic peripheral blood leukocytes (PBLs) can be used.

The magnetic peripheral blood leukocyte cells used as the support cells are inactivated to ensure safety. As the method of inactivation, a conventional method known in the art may be used, and for example, a method of irradiating gamma-rays may be used. Such inactivated feeder cells include isolated T-cells.

Proliferation method using a support cell as in the present invention is a method of purely separating the natural killer cells and then proliferating, there is an advantage that can continue to grow only pure natural killer cells thereafter.

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

A CD3 antibody is a protein that specifically reacts with a CD3 antigen, a group of molecules that associate with a T cell receptor (TCR) to form an antigen recognition complex. CD3 molecules have a longer intracellular region and a antigen recognition signal in the cell compared to TCR. It is in charge of conveying.

Examples of anti-CD3 antibodies that can be used in the present invention include OKT-3, UCHT1, HIT3a Etc., Preferably it is an OKT-3 antibody.

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

As an example of the interleukin protein which can be used by this invention, IL-2, IL-15, IL-12, IL-18, IL-21, etc. are mentioned, Preferably it is an IL-2 protein.

In the culture method of the present invention, NK cells and PBLs isolated from human peripheral blood are added to a culture medium for normal animal cell culture such as A IM-V media, RIMI1640, CellGro SCGM, and X-VIVO20. And interleukin protein is incubated. In one embodiment of the present invention was cultured by the addition of the OKT-3 antibody and IL-2. The concentration of the OKT-3 antibody to be added is 0.1-100 ng / ml, preferably about 10 ng / ml, and the concentration of IL-2 is 10-2000 U / ml, preferably about 500 U / ml. .

It may also be cultured by adding an additional growth factor that supports the growth of serum or plasma and lymphocytes. The kind of serum or plasma added to the medium is not particularly limited, and commercially available ones derived from various animals can be used, but those derived from humans are more preferable as human origin. For example, a method known to those skilled in the art can be used, such as a combination of cytokines for propagating lymphocytes from PBMCs, lectins for stimulating lymphocyte proliferation, and the like.

In another aspect, the present invention provides an optimal culture concentration of NK cells capable of significantly proliferating NK cells.

As described above, one aspect of the present invention will be described in more detail, comprising: first culturing the isolated natural killer cells in the presence of peripheral blood leukocyte cells in a medium containing anti-CD3 antibody and interleukin protein; Removing anti-CD3 antibodies from the culture; And further culturing the culture medium from which the anti-CD3 antibody has been removed by adding it to a medium containing interleukin protein.

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

Preferably, natural killer cells are inoculated at a concentration of 1 × 10 ⁵ to 1 × 10 ⁶ cells / well, more preferably 1 × 10 ⁵ to 3 × 10 ⁶ cells / well. In particular, when inoculated at a concentration of 2 x 10 ⁵ cells / well, it was confirmed through experiments to show a growth rate of about 900 times 14 days after incubation.

In the present invention, natural killer cells are treated with anti-CD3 antibody such as OKT-3 antibody and interleukin protein such as IL-2 at the appropriate concentration while using support cells, thereby using only support cells or only OKT-3 antibody stimulation. Compared to the previous studies used, high-purity natural killer cells can be expanded more explosively in a short time.

In another aspect, the present invention relates to natural killer cells obtained by the above method. The surface type characteristics of the natural killer cells proliferated and cultured according to the above method are described below.

More than 90% of the normal NK cells isolated from normal peripheral blood have a surface type of CD3- / CD56 +. When the culture is proliferated according to the method of the present invention, CD3 + T cells are relatively reduced and more CD3- / CD56 + NK cells are observed at 7 days, and almost all CD3 + T cells are disappeared at about 10% from the day of culture, and more than 95% of cells are present. All are activated NK cells expressing CD16. In other words, high-purity natural killer cells having a surface type of CD16 + can be obtained by proliferating in a short time.

Therefore, using a large amount of activated NK cells, which can be clinically applied, a cell therapy agent can be prepared for tumor treatment or removal of virus-infected cells that are supposed to be the source of tumors.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example  1: supporting cells ( Feeder cell ) Preparation and natural killer cells ( NK cell ) Separation

(1) Feeder cell preparation

20 ml of peripheral blood of a normal person was drawn, and 5 ml of blood was placed in a 15 ml conical tube. 5 ml of saline was added to the blood and mixed evenly with a pipette. Add 5 ml of ficoll (GE healthcare, Uppsala, 17-1440-03) to a new 15 ml conical tube, carefully place the mixed (diluted) blood in a 15 ml tube containing picol, and then 2000 rpm. Centrifuged at room temperature for 30 minutes (Hanil, Korea, Union32-R).

After taking the immune cell layer formed between the picol and plasma (plasma), transfer to a new 15 ml conical tube, filled with HBSS up to 10 ml, evenly mixed the cells and centrifuged for 10 minutes at 1200 rpm. The supernatant was removed by vacuum suction. Again 10 ml of HBSS was added and the process of centrifugation was repeated.

After 1 ml of AIM-V media (Invitrogen, 12055091) containing 5% of the cell culture solution (hAB serum (Sigma, H4522)) was released, 10 ul of the cell solution was transferred to a microtube and trypan blue (trypan). blue) (Gibco) 90 ul, mix well with a pipette, stain with trypan blue (Gibco, 15250-061) dye, and count the cells using an inverted microscope (Olympus, CK2-TRC-2). Cell culture medium was diluted to 5 x 10 ^ 6 cell / ml.

Transfer 1 ml of cells into 5 ml tubes for FACS analysis, and rest cells inactivated by gamma radiation of 2000cGy (gamma-irradiatior, MDS Nordion, Gammacell 3000 Elan). Was prepared.

(2) NK cell separation

15 ml of blood collected from a normal person was transferred to a new 50 ml conical tube, and 750 ul of Rosettesep NK cell enrichment cocktail (Stemcell technologies, 15065) was added, followed by a slow rotation at room temperature for 20 minutes. 15 ml physiological saline was added to the finished blood and mixed well.

Add 5 ml of each picol to 3 new 15 ml conical tubes, carefully add 10 ml of blood containing the physiological saline solution to the 15 ml conical tube containing the picol, and centrifuge at room temperature for 30 minutes at 2000 rpm. It was. At the end of the centrifugation, the natural killer cell layer between Picol and autologous plasma solution was taken and transferred to a new 15 ml conical tube, filled with HBSS to 10 ml, and further centrifuged at 1500 rpm for 10 minutes. After removing the supernatant, and filled with HBSS up to 10 ml again, the cells were released well, centrifuged for 10 minutes at 1200rpm. The supernatant was removed by vacuum, and 1 ml of cell culture was added to free the cells.

Transfer 10 ul of the cell diluent to the microtube, mix 40 ul of trypan blue and pipette well, stain with trypan blue, and measure the cell number, using cell culture solution to make the cell number 1 x 10 ⁶ cells / ml. Diluted.

(3) Characteristics of isolated initial NK-cell

Surface morphology was analyzed by staining anti-human CD3-FITC and anti-human CD56-APC antibodies on the isolated NK cells. As a result, as can be seen in Figure 1 it was confirmed that more than 90% of the initially isolated cells are NK cells of CD3- / CD56 +.

Example  2: Cultivation of Isolated Natural Killer Cells

Initial cells were cultured in 12-well plates (Falcon). 500 ul of the supporting cells prepared in Example 1- (1) was put in the well, and 500 ul of the natural killer cells isolated in Example 1- (2) was further added to the well containing the supporting cells.

Add 10 ng / ml of IL-2 (Norvatis) cytokine and OKT-3 antibody (ebioscience, 16-0037) at 500 U / ml to each well containing the cells, and shake the plate carefully to make sure the cells and cytokines Mixed evenly.

Plates were incubated for 5 days in a 37 degree Celsius wet incubator containing 5% carbon dioxide, at which time no culture or cytokine was added.

On day 5 from the day of culture, cells were collected in 15 ml conical tubes by pipetting all wells containing cells. 1 ml of each cell culture was added to each well from which cells were removed, and the remaining cells were collected. The collected cells were centrifuged at 1200 rpm for 10 minutes. The supernatant was vacuum suctioned to remove the OKT-3 antibody.

The remaining cells were diluted with 2 ml of cell culture solution, 10 ul of diluted cells were taken into a microtube, mixed well with 90 ul of trypan blue solution, and stained with trypanblue to measure the number of cells. Cell culture medium was added and diluted to 2 x 10 ⁵ cells / well.

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

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

On the 10th day after the first incubation, cells were harvested and counted. The harvested cells were replanted in 1 × 10 ⁷ cells in a T75 flask and 5 ml of cell culture medium containing 500 U / ml of IL-2. Put them in. The cell culture was added 5 ml daily until transferred to the next flask.

When the cells were no longer able to proliferate, the cells were transferred to a new flak, and 5 ml of the cell culture solution containing IL-2 (500 U / ml) was added every day until the 17th day.

Example  3: obtained NK - cell Surface analysis

After removal of OKT-3, only IL-2 was treated alone and some cells were harvested on day 7 and day 10 to analyze the surface type.

Cells were harvested before, during or after the culture, and centrifuged at 12000 rpm for 5 minutes, and the culture solution was removed by suction. Cell number was measured by diluting with 1 ml FACS buffer (2.5% FBS + PBS), and diluted with FACS buffer to 5 x 10 ⁶ cells / ml. 100 ul of the diluted cell solution was added to the FACS tube (Falcon), and the antibody was added as follows.

     Tube 1: Un-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 tubes were left to stand at refrigeration temperature for 30 minutes and stained. Then, 2 ml FACS buffer was added to the stained cells and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and again, 2 ml FACS buffer was added and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed again, and 300 ul FACS buffer was added and vortexed to release the cells. Surface type was analyzed using FACSCalibur (Becton Dickinson).

As a result, as can be seen in Figure 2, when treated with OKT-3 on day 7, the CD3 + T cells were relatively reduced and more CD3-CD56 + NK cells than the culture without treatment. CD3 + cells were assumed to be irradiated PBMCs that have not yet died. On the 10th day from the culture day, almost all CD3 + T cells disappeared when cultured using both OKT-3 and feeder PBMC, and more than 95% of the cells were confirmed to be NK cells. That is, all of the proliferated NK cells were activated NK cells expressing CD16.

Example  4: cultured NK - cell of Cell killing ability  evaluation( Cr - release assay )

(1) Effector cell preparation

On day 14 of natural killer cell culture, some cells were collected, centrifuged at 1200 rpm for 5 minutes, the supernatant was removed, and then diluted with 2 ml cell culture solution. After diluting by adding the cell culture solution to 3 x 10 ⁶ cells / ml, the effector: Target cell (E: T) ratio was adjusted to 30: 1.

1 ml of the prepared cells were taken into a new tube, and 2 ml of the cell culture solution was added and mixed well to adjust Effector: Target cell (E: T) ratio = 10: 1. In addition, 500 ul of cells were taken and placed in a new tube, and the mixture was added to the culture medium and further mixed with 4.5 ml, and Effector: Target cell (E: T) ratio was adjusted to 3: 1.

100 ul of natural killer cells adjusted to the above specific ratio were added to a 96-well plate to achieve 3 wells / ratio for each target.

(2) Target cell preparation

Prepare the acute lymphoblastic leukemia cell line CEM and chronic myelogenous leukemia (CML) cell line, which are 80% confluency, and collect the two cell lines, place them in a 15 ml conical tube and centrifuge at 1200 rpm for 5 minutes. Separated. Supernatant was removed and cells were diluted in 5 ml cell culture. The cell number was measured and transferred to a new 15 ml tube by 1 × 10 ⁶ cells. Cell culture solution was added to the transferred cells to 10 ml, and then centrifuged at 1200 rpm for 5 minutes. The supernatant was removed, 25 μl of FBS was added to dilute cells, and 100 μl of Cr-51 (Perkin Elmer) was added.

After the tube was placed in a 37 ° C. wet incubator containing 5% carbon dioxide and left for 1 hour, the cells were taken out, filled with the cell culture solution to 10 ml, and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed and washed twice more in the same manner. After washing the cells, 10 ml of the cell culture solution was evenly diluted with a pipette.

(3) killing ability measurement

The diluted cell line was added 100 ul per target to the U-bottom 96-well plate (FALCON) containing the previously prepared effector cells. As spontaneous control for each target, 100ul of the target cell was placed in three wells without effector cells, and 100ul of the cell culture solution was added. As a maximum control for each target, 100ul of target cell was put in three wells without effector cells, and 100ul of PBS containing 1% triton X-100 was incubated for 4 hours.

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

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

Example  5: cultured NK - cell Cells Proliferative capacity  evaluation( CFSE - proliferation assay )

As in Example 1, NK cells were isolated from the peripheral blood of 15 ml using Rossetsep, and autologous peripheral blood cells were irradiated to inhibit proliferation, and then used as feeder cells. Anti-CD3 antibody (OKT-3) was stimulated with PBL for 5 days at low concentration and then cultured for 17 days in media containing IL-2 to confirm up to 600-fold NK cell proliferation.

As a result, as can be seen in Figure 4, if there is no stimulation of the OKT-3 antibody, it is only 20-fold proliferation after 10 days of cultivation, while 112 times after 10 days of cultivation with OKT-3 stimulation, the maximum after 17 days of culture A 600-fold proliferation was confirmed.

Comparative example  1: cultured NK - cell Cells Proliferative capacity  compare

In order to compare the evaluation of NK-cell proliferation capacity, each was cultured under the following conditions.

  a. NK cell + IL-2 (500 U / ml)

  b. NK cell + IL-2 (500U / ml) + OKT-3 (10 ng / ml)

  c. NK cell + IL-2 (500 U / ml) + irradiated PBMC

  d. NK cell + 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 day, each cell was harvested and the number of cells was measured.

1 x 10 ⁶ cells were prepared for each of the above conditions, transferred to a 5 ml tube, the cell culture was filled to a final 500 ul, and 5 μM of CFSE solution was added thereto, followed by standing in a cell incubator for 30 minutes. After washing three times with PBS, 530 nm wavelength was analyzed using FACS Calibure.

As a result, as can be seen in Figure 5, culturing with a feeder than culturing only NK cells alone promotes cell proliferation, and treatment with OKT-3 with a feeder is much more effective for cell proliferation Confirmed. In addition, the most remarkable effect was the simultaneous treatment of IL-2 and OKT-3, which is the d condition, showed about 200-fold proliferation in 14-day culture and about 600-fold proliferation in 17-day culture.

Example  7: cultured NK - cell Cells by concentration Proliferative capacity  evaluation

As in Example 2, the initial culture and OKT-3 removal were performed in the same manner, and when the number of NK-cells was measured, the cells were diluted at different concentrations as follows, and seeded in a 12-well plate, and 9 in a cell incubator. The cells were further cultured for days, and the number of cells was measured.

a. 1 x 10 ⁵ cells / ml / well

b. 2 x 10 ⁵ cells / ml / well

c. 5 x 10 ⁵ cells / ml / well

d. 1 x 10 ⁶ cells / ml / well

As a result of comparing the cell proliferation according to each condition, as shown in Figure 6, when seeding at 2 x 10 ⁵ cell / ml showed a maximum proliferation about 900 times in 14 days, 1 x 10 ⁶ cells / ml In case of seeding, the growth rate was lowest at about 100 times at 14 days. Therefore, after removing OKT-3, it was confirmed that the seeding concentration acts as a very large factor in natural killer cell proliferation.

Having described the contents of the present invention in detail, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. . Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a result of analyzing the surface type of the initial NK cells isolated from the peripheral blood of normal people.

Figure 2 shows the results of analyzing the surface type of NK-cell obtained by culturing according to the method of the present invention.

Figure 3 is the result confirming the tumor killing ability of NK-cell obtained by culturing according to the method of the present invention.

4 is a result confirming the proliferation capacity of the NK-cell obtained by culturing according to the method of the present invention.

5 is a result of comparing the proliferative capacity of NK-cells obtained by culturing in each culture condition according to the presence or absence of support cells PBMC, anti-CD3 antibody OKT-3 and IL-2.

Figure 6 is a result of comparing the proliferation capacity of NK-cell obtained by culturing by varying the seeding (seeding) concentration of natural killer cells.

Claims (12)

A method of propagating natural killer cells, comprising culturing the natural killer cells in the presence of anti-CD3 antibody and interleukin protein in the presence of peripheral blood leukocyte cells. The method of claim 1, wherein the anti-CD3 antibody is selected from the group consisting of OKT3, UCHT1 and HIT3a. The method of claim 2, wherein the anti-CD3 antibody is an OKT-3 antibody. The method of claim 1, wherein the interleukin protein is selected from the group consisting of IL-2, IL-15, IL-12, IL-18 and IL-21. The method of claim 4, wherein the interleukin protein is interleukin-2 (IL-2). The method of claim 1, wherein the peripheral blood leukocyte cells are inactivated. The method of claim 6, wherein the inactivated peripheral blood leukocytes are purified T cells. The method of claim 1, wherein the peripheral blood leukocyte cells are autologous peripheral blood leukocyte cells. The method of claim 1, Culturing the natural killer cells in the presence of anti-CD3 antibody and interleukin protein in the presence of peripheral blood leukocyte cells, Removing an anti-CD3 antibody from the culture, and The method of proliferating natural killer cells comprising the step of adding the culture medium from which the anti-CD3 antibody is removed to the medium containing the interleukin protein and further culture. The method of claim 9, wherein the anti-CD3 antibody is an OKT-3 antibody, and the interleukin protein is interleukin-2 (IL-2). 10. The method of claim 9, wherein the culture medium from which the anti-CD3 antibody is removed is added at a concentration of 1 x 10 ⁵ to 1 x 10 ⁶ cells / well. The method of claim 11, wherein the culture medium from which the anti-CD3 antibody is removed is added at a concentration of 1 × 10 ⁵ to 3 × 10 ⁶ cells / well.

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