WO2022060056A1 - Method for preparing high purity natural killer cells with high efficiency, and use thereof - Google Patents

Abstract

The present invention relates to a method for preparing high purity natural killer cells (NK cells) with high efficiency and, more specifically, to a composition for enhancing the proliferation and activation of NK cells and a method for preparing the NK cells having enhanced proliferation and activation. An NK cell preparation technique according to the present invention enables high purity NK cells to be obtained with high efficiency, and can overcome limitations of a conventional technique using irradiated cancer cells as feeder cells, and thus the NK cells prepared by the method according to the present invention can be effectively used in the field of treating diseases such as cancer and autoimmune diseases on the basis of high purity and excellent cancer-cell-killing ability.

Description

High-purity and high-efficiency natural killer cell manufacturing method and use thereof

The present invention relates to a method for producing high-purity and high-efficiency Natural Killer cells (NK cells), and more particularly, to a composition for enhancing the proliferation and activity of NK cells, and the production of NK cells with enhanced proliferation and activity it's about how

In cancer patients, the treatment process using chemotherapy has a side effect that the patient's immune cells decrease and the immune function decreases. Therefore, a treatment technology that activates the reduced number and function of immune cells by transplanting immune cells that have been proliferated and activated outside the body into the body of a patient can have a synergistic effect when combined with the existing anti-cancer therapy, It is possible to prevent cancer recurrence because it has a removal effect on cancer and micro-residual cancer.

Natural killer cells (NK cells) used in immune cell therapy are morphologically large cells with large granules in the cytoplasm and account for about 5 to 15% of lymphocytes in the blood. NK cells are differentiated from common lymphoid progenitors like T cells or B cells, and are cytotoxic lymphocytes that play a role in eliminating tumor cells or virus-infected cells. However, NK cells can recognize cancer cells by themselves without an activation process such as antigen presentation, differentially from existing B cells, T cells, and dendritic cells (DC cells), granzyme and perforin, etc. It can directly kill cancer cells by using the enzyme of It has been reported that such defects in differentiation and activity of NK cells are related to various carcinomas such as breast cancer, melanoma cancer, and lung cancer, and the excellent killing ability of NK cells is the treatment of solid cancers and infectious diseases and rejection of bone marrow or organ transplantation. It can be applied to a new immune cell therapy to prevent a reaction.

However, despite the potential of NK cells as a therapeutic agent for various diseases as described above, the number of cells present in the body is not large, so it is most important to secure a large number of NK cells for effective use in cell therapy. However, the number of NK cells in normal people is small, and especially in cancer patients, the number, differentiation and function of NK cells are lowered, so it is difficult to secure a sufficient number of cells for use as a therapeutic agent, and mass proliferation in vitro . and culture is not easy.

In this regard, various studies have been made on culture techniques for amplifying and activating NK cells to a practically useful level. For example, it has been reported that conventionally, NK cell culture technology using IL-2 or other cytokines and compounds did not dramatically increase the number of NK cells compared to the initial stage, and cancer cells irradiated as a culture technology using support cells, Results of effectively improving the proliferation of NK cells using K562 cells transformed to express MICA, 4-1BBL, and IL-15 have been reported (Tissue Antigens, 76(6): p467-475, 2010), but these All of the technologies used cancer cells have limitations in that they used methods that were not suitable for ensuring important safety in clinical applications, such as the possibility that the cancer cells could be mixed with cell therapy preparations.

Therefore, there is an urgent need for a method for obtaining NK cells with high purity and high efficiency for securing a large amount of NK cells that can overcome these conventional problems while maintaining sufficient cancer cell killing ability.

The present inventors overcame the above conventional problems and as a result of intensive research to establish an optimized NK cell production technology capable of obtaining NK cells with high purity and high efficiency, culturing for the purpose of activating and proliferating NK cells in the blood We have developed a method for producing NK cells with markedly enhanced proliferation and activity only by adding CD antibodies and cytokines without the use of support cells such as cancer cells, including a process for dividing, thus completing the present invention.

Accordingly, an object of the present invention is to provide a composition for enhancing the proliferation and activity of natural killer cells (NK cells).

Another object of the present invention is to provide a method for producing natural killer cells (NK cells) with enhanced proliferation and activity.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides interleukin-2 (Interleukin-2), interleukin-12 (Interleukin-12) and an anti-CD16 antibody comprising as an active ingredient, natural killer cells (Natural killer) cells) to provide a composition for promoting proliferation and activity.

In one embodiment of the present invention, the composition further comprises one or more selected from the group consisting of interleukin-15, interleukin-18, anti-CD3 antibody and anti-CD56 antibody. may be doing

In another embodiment of the present invention, the composition may further include autologous serum.

In addition, the present invention comprises the steps of (a) isolating monocytes from the peripheral blood of mammals including humans; (b) putting the mononuclear cells and the culture solution into a culture flask coated with at least one selected from the group consisting of anti-CD3 antibody, anti-CD16 antibody and anti-CD56 antibody, and then primary culturing for 5 to 7 days; And (c) recovering the cultured cells and a secondary culture for 5 to 7 days with the culture medium, as a method for producing natural killer cells (Natural killer cells), the culture medium interleukin-2 (Interleukin- 2), interleukin-12 (Interleukin-12), interleukin-15 (Interleukin-15) and interleukin-18 (Interleukin-18) characterized in that it contains one or more selected from the group consisting of, and autologous serum, A manufacturing method is provided.

In one embodiment of the present invention, the autologous serum may be added in an amount of 5 to 15%.

In addition, the present invention provides a natural killer cell (Natural killer cells) prepared by the above method.

In addition, the present invention provides a cell therapy agent for cancer treatment comprising the natural killer cells as an active ingredient.

In addition, the present invention provides a cell therapy agent for the treatment of infectious diseases, comprising the natural killer cells as an active ingredient.

In addition, the present invention provides a method for preventing or treating cancer or an infectious disease comprising administering the natural killer cells to an individual in need thereof.

In addition, the present invention provides the use of the natural killer cells for the manufacture of a medicament for the prevention or treatment of cancer or infectious disease.

The NK cell production technology according to the present invention can obtain high-purity and high-efficiency NK cells by significantly improving the proliferation and activity of NK cells, and overcome the limitations of the prior art using cancer cells as support cells. The NK cells prepared by the method according to the present invention may be usefully used in the treatment of various related diseases, such as cancer, infectious diseases, and autoimmune diseases, based on their high purity and excellent ability to kill cancer cells.

1 is a result of FACS analysis to find out the ratio of NK cells to the culture obtained after the primary activation culture and secondary proliferation culture in the NK cell manufacturing process according to the present invention.

2 is a result of analyzing the cancer cell killing rate of NK cells obtained after primary activation culture and secondary proliferation culture in the NK cell manufacturing process according to the present invention.

3 is a result showing microscopic images of NK cells activated in the primary activation culture and secondary proliferation culture in the NK cell manufacturing process according to the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors overcame the limitations of the conventional NK cell production technology and as a result of intensive research to establish an optimized method for producing NK cells capable of obtaining NK cells with high purity and high efficiency, proliferation only with the addition of CD antibodies and cytokines And a method for preparing NK cells with enhanced activity was developed, thereby completing the present invention.

Accordingly, the present invention provides a composition for enhancing the proliferation and activity of natural killer cells, comprising interleukin-2, interleukin-12, and an anti-CD16 antibody as an active ingredient to provide.

As used herein, the term “cell proliferation” means that cells undergo a series of cell division steps to increase or differentiate the number of cells in culture. In the present invention, “proliferation of natural killer cells (NK cells)” refers to an increase in the number of NK cells in culture after NK cells undergo a cell division stage or an increase in the number of cells by differentiation from immature blood cells to NK cells. .

As used herein, the term “enhancement of natural killer cells (NK cells) activity” refers to tumor cells or virus-infected cells in culture when immature NK cells become mature NK cells or inactive NK cells are activated. It includes both an increase in the number of cells having a cell killing ability to remove, or an enhancement in cell killing efficiency of individual NK cells.

In the present invention, NK cells, which are raw materials for promoting proliferation and activity, may be purchased commercially or may be collected from humans or animals, and preferably may be supplied from humans in need of treatment with NK cells.

In addition, the NK cells may be isolated from any tissue source in vivo, preferably contained in blood collected from the living body, more preferably whole blood, umbilical cord blood, bone marrow or peripheral blood, most preferably Alternatively, it can be isolated from peripheral blood.

In the present invention, the composition may further include one or more selected from the group consisting of interleukin-15, interleukin-18, anti-CD3 antibody and anti-CD56 antibody. there is.

The cytokine used in the present invention is a type of interleukin, and interleukin is a generic term for proteinaceous biologically active substances produced by immune cells such as lymphocytes, monocytes and macrophages. In the present invention, it is selected from the group consisting of interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15) and interleukin-18 (IL-18), in particular IL -2 and IL-12 may be used, and preferably, all of the above IL-2, IL-12, IL-15 and IL-18 may be used.

The anti-CD antibody used in the present invention is selected from the group consisting of an anti-CD16 antibody, an anti-CD3 antibody, and an anti-CD56 antibody. In particular, an anti-CD16 antibody may be used, preferably the anti-CD16 antibody , both anti-CD3 antibodies and anti-CD56 antibodies can be used.

The composition for enhancing proliferation and activity of NK cells of the present invention further comprises other essential components or other carriers, or adjuvants, including autologous serum, in order to promote proliferation and activity of NK cells, in addition to the interleukin and anti-CD antibody can do.

In another aspect of the present invention, the present invention provides a method comprising: (a) isolating monocytes from the peripheral blood of a mammal, including a human; (b) putting the mononuclear cells and the culture solution into a culture flask coated with at least one selected from the group consisting of anti-CD3 antibody, anti-CD16 antibody and anti-CD56 antibody, and then primary culturing for 5 to 7 days; And (c) recovering the cultured cells and a secondary culture for 5 to 7 days with the culture medium, as a method for producing natural killer cells (Natural killer cells), the culture medium interleukin-2 (Interleukin- 2), interleukin-12 (Interleukin-12), interleukin-15 (Interleukin-15) and interleukin-18 (Interleukin-18) characterized in that it contains one or more selected from the group consisting of, and autologous serum, A manufacturing method is provided.

In the step (a) of the present invention, the peripheral blood mononuclear cells (PBMC) are isolated from the peripheral blood of a mammal, preferably a human, mainly B cells, T cells, immune cells such as natural killer cells, and basophils (basophil), eosinophils (eosinophil), and includes granulocytes such as neutrophils (neutrophil). The PBMC may be prepared by a conventional method from peripheral blood collected in vivo. For example, in the present invention, it was separated from peripheral blood using a specific gravity centrifugation method using Ficoll.

In addition, the peripheral blood mononuclear cells may be obtained from a subject in need of treatment, that is, autologous peripheral blood mononuclear cells. When the peripheral blood mononuclear cells are autologous, even if some T cells are present in the proliferated NK cell population, there is an advantage in that it is not necessary to remove the T cells because all the cells are derived from the patient.

The step (b) of the present invention is a culture step for activating NK cells in peripheral blood mononuclear cells.

In the step (b) of the present invention, in the method of coating the anti-CD antibody on the culture flask, a coating solution in which DPBS and the anti-CD3 antibody, anti-CD16 antibody and anti-CD56 antibody are mixed is placed in a culture flask and evenly distributed. After dispersing, it can be carried out through a process of removing the coating solution by leaving it to stand for 2 hours to 6 hours, preferably 4 hours, or 16 hours to 24 hours, preferably 18 hours in refrigeration conditions at 37 ° C. and then washing. .

After putting the monocytes and culture solution isolated in step (a) into the coated culture flask, it can be cultured for 5 to 7 days, preferably for 6 days, but in order to obtain NK cells desired by those skilled in the art, It can be appropriately set within the range of the number of incubation days. The number of inoculated cells is preferably 1 x 10 ⁶ cells or more based on the T75 flask.

In a specific embodiment of the present invention, as a result of the primary culture in step (b), an increase in the number of cells, a high cell viability of at least 87.6% or more, was confirmed, and the ratio of NK cells increased from an average of 13.9% to 47.1%. When the activation culture period was increased by 1 day, it was confirmed that the ratio of NK cells increased by 15% on average.

The step (c) of the present invention is a proliferation culture step to significantly increase the number of NK cells.

The step (c) of the present invention may be accomplished by recovering the cells cultured through step (b) and culturing for 5 to 7 days using a culture medium of the same composition as in step (b), preferably 6 It can be cultured for one day, but one skilled in the art can appropriately set within the range of the number of culture days to obtain the desired NK cells.

In a specific embodiment of the present invention, as a result of the secondary culture in step (c), NK cells out of about 3 x 10 ⁹ cells obtained account for a very high rate of 94.42% and show a high survival rate of 93.2%. It was confirmed that the thymoma cell line, K562 cells, showed an average cancer cell killing rate of 70%.

In the steps (b) and (c), the culture conditions are conventional cell culture conditions, that is, about 37° C., CO ₂ It may be performed in an incubator. As the culture vessel, a culture flask, more preferably a T75 flask, is used in the present invention, but a person skilled in the art may appropriately select and apply from commercially available culture dishes, flasks, plates, multi-well plates, and culture bags.

In the present invention, as the basal medium added to the culture medium for the production of NK cells, an animal cell culture medium commonly used in the art such as RPMI1640 medium, CellGro medium, AIM-V medium and XVIVO 20 can be used. In the present invention, RPMI1640 medium was used, but the type of medium can be appropriately selected and applied by those skilled in the art. In addition, the culture medium may further contain other components necessary for proliferation and culture of NK cells in addition to the additives described above, if necessary.

In the present invention, autologous serum additionally added to the culture medium may be added in an amount of 5 to 15%, preferably 7 to 12 v/v%, more preferably 9 to 11 v/v%, most Preferably, it may be added at 10 v/v%.

The present invention has specifically demonstrated that NK cells with high purity and high efficiency can be obtained through the method for preparing NK cells compared to PBMCs through Examples.

As another aspect of the present invention, the present invention provides a natural killer cell (Natural killer cells) prepared by the above method.

In addition, the present invention provides a cell therapy agent for cancer treatment comprising the natural killer cells as an active ingredient.

The cancer may include all types of cancer including solid cancer and hematological cancer. Unlike blood cancer, solid cancer refers to cancer formed by forming a lump in an organ, and cancer occurring in most organs corresponds to this. There is no particular limitation on carcinomas that can be treated using the NK cells according to the present invention, and gastric cancer, liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, and the like.

In addition, the present invention provides a cell therapy agent for the treatment of infectious diseases, comprising the natural killer cells as an active ingredient.

The infectious disease is a disease caused by infection with a virus or a pathogen, and is a concept including all diseases that can be transmitted through respiratory, blood, and skin contact, and the like.

Non-limiting examples of such infectious diseases may include hepatitis B, hepatitis C, human papilloma virus (HPV) infection, cytomegalovirus infection, viral respiratory disease and influenza.

The term "treatment" may refer to any action in which the symptoms of cancer or infectious disease of an individual are improved or changed advantageously by the administration of the natural killer cells.

The cell therapy product may be provided as a cell therapy product including an active ingredient alone, or one or more pharmaceutically acceptable carriers, excipients or diluents.

Specifically, the carrier may be, for example, colloidal suspension, powder, saline, lipid, liposome, microspheres or nano-spherical particles. They may form complexes with or be associated with a vehicle and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.

When the cell therapy product is formulated, it may be prepared using a diluent or excipient such as a suspending agent, preservative, or filler commonly used. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, and emulsions. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. In addition, for topical administration, combining organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers and chemical derivatives thereof, etc. may include When the cell therapy agent is prepared in a formulation suitable for injection, the natural killer cells may be dissolved in a pharmaceutically acceptable carrier or frozen in a lysed solution. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. The cell therapy agent is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. It may be prepared by introduction into a dose container.

The term "administration" means introducing a predetermined substance into a subject by an appropriate method, and "subject" means any living organism, including humans, mice, mice, livestock, etc., capable of carrying cancer or an infectious disease. As a specific example, it may be a mammal including a human.

The cell therapy agent may be administered parenterally, and when administered parenterally, external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection Injection, transdermal injection, intranasal injection, enteral injection, local injection, sublingual injection, rectal injection, or intrathoracic injection injection method can be selected.

The cell therapy agent is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type, severity, activity of the drug, and the drug in the patient. It can be determined according to factors including sensitivity, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. Based on an adult patient weighing 70 kg, for example, about 1,000-10,000 cells/time, 1,000-100,000 cells/time, 1,000-1000,000 cells/time, 1,000-10,000,000, 1,000-100,000,000 cells/time, 1,000 to 1,000,000,000 cells/time, 1,000 to 10,000,000,000 cells/time, or 1,000 to 100,000,000,000 cells/time, may be dividedly administered once or several times a day at regular time intervals, or may be administered several times at regular time intervals.

In addition, the present invention provides a method for preventing or treating cancer or an infectious disease comprising administering the natural killer cells to an individual in need thereof.

The term "prevention" may refer to any action of suppressing or delaying the onset of cancer or infectious disease in an individual by the administration of the natural killer cells.

In addition, the present invention provides the use of the natural killer cells for the manufacture of a medicament for the prevention or treatment of cancer or infectious disease.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. High-purity and high-efficiency NK cell production method

The present inventors conducted an experiment according to the following procedure to prepare NK cells with high purity and high efficiency.

1) Collection of autologous blood

After collecting blood from the human body, it was prepared for blood separation by putting it in sodium heparin tube and plain tube.

2) Coating the culture flask

A coating solution was prepared by mixing 13 ml of DPBS and 1 ml of a mixture of CD3, CD16 and CD56 in a 50 ml tube. Then, 14 ml of the coating solution was placed in a T75 flask and evenly dispersed over the entire area. After standing at 37° C. for 4 hours or refrigerated for 18 hours, the coating solution was removed, 10 ml of DPBS was added, and washed once to prepare.

3) Serum Preparation

In the blood, the plain tube blood was centrifuged at 2000rpm at 4°C for 30 minutes. After centrifugation, the supernatant was recovered, filtered using a 0.2um membrane filter, and refrigerated for use in culture.

4) Immune cell isolation

The blood from the Heparin tube was transferred to the BSC, transferred to a 50ml tube, and the total amount was checked, and then the same amount of DPBS was added and mixed. The same amount of Ficoll as the blood was transferred to a 50ml tube, and then a mixture of DPBS and blood was added to the tube containing Ficoll to prepare sufficiently. Next, the prepared tube was centrifuged at 2000 rpm at 4° C. for 20 minutes, and after centrifugation was completed, the buffy coat layer was recovered and transferred to a new 50 ml tube. DPBS of twice the volume of the recovered buffy coat was added and centrifuged at 1200 rpm for 5 minutes at 4°C. After centrifugation, the supernatant was removed, the cells were well released, and 20 ml of double volume DPBS was added, followed by centrifugation at 1200 rpm for 5 minutes at 4°C. After removing the supernatant and releasing the cells, the culture solution was added to resuspend the cells, and the number of cells was measured.

5) Activated culture of immune cells

IL-2, IL-12, IL-15, IL-18 and autologous serum were added to 50 ml of RPMI culture medium. The prepared culture solution was placed in a T75 flask, and immune cells of 1,000,000 cells or more were seeded. Cells were activated and cultured for 6 days in an incubator at 37° C., 5% CO ₂ .

6) Immune cell proliferation and culture

After the activation culture for 5-7 days, all cultures were recovered in a 50ml tube and centrifuged at 1200rpm, 5min, 4℃. Then, the supernatant was removed, the cells were released, and the culture solution was added to resuspend the cells, and the number of cells was measured. The cells were suspended so that the total amount of the RPMI culture medium supplemented with IL-2, IL-12, IL-15, and IL-18 was 200 ml, and seeded in 1000 ml polyvinyl culture medium. At this time, 4% autologous serum was added. Cells were cultured at 37° C., 5% CO ₂ in an incubator for 6 days.

7) Addition of immune cell medium

After 3 to 5 days, when the culture medium turns yellow and colonies are formed, the cell number and viability are measured, and the culture medium is added to the RPMI medium containing IL-2, IL-12, IL-15, and IL-18 so that the total volume becomes 1000 ml. added.

8) Recovery and charging of immune cells

After the culture was transferred to 4 250ml tubes, the supernatant was discarded and centrifuged at 1200rpm, 10 minutes, 4℃. Thereafter, the supernatant was discarded, the cells were well released, and the cells were suspended in 100 ml of normal saline to measure the cell number and viability.

Example 2. Analysis of Activation Culture Results-Cell Number/Viability/NK Cell Ratio

The present inventors prepared NK cells with enhanced proliferation and activity according to the procedure of Example 1. To this end, first, blood was collected from four donors and the characteristics of PBMCs in the blood were analyzed, and the results are shown in Table 1 below.

blood volume number of cells survival rate NK ratio N200515-01TS 188 1.43 x 10 ⁸ 92.5 17.0 N200515-02TS 184 1.62 x 10 ⁸ 93.9 10.8 N200612-01TS 112 9.4 x 10 ⁷ 89.3 1.6 N200612-02TS 101 1.04 x 10 ⁸ 92.7 16.9 Average 146 1.04 x 10 ⁸ 92.1 13.9

Next, PBMCs in the blood of each donor were subjected to activation culture according to the method described in Example 1 with the number of inoculated cells of 2 x 10^7/50ml and the inoculated cell density of 4 x 10^5 /ml. , the cell number, viability, and ratio of NK cells after culture were analyzed.

First, as a result of cell number analysis, as shown in Table 2 below, an average of 10.0x10^7 cells were recovered on the 6th day of culture, and at least 8.23x10^7 cells could be obtained.

6 days N200515-01TS 8.23E+07 N200515-02TS 1.11E+08 N200612-01TS 1.03E+08 N200612-02TS 1.05E+08 Average 1.00E+08 Standard Deviation 1.25E+07

Next, as a result of measuring the cell viability, as shown in Table 3 below, the average cell viability was 88.85% on the 6th day, indicating a survival rate of at least 87.6%.

6 days N200515-01TS 89.9 N200515-02TS 88.7 N200612-01TS 89.2 N200612-02TS 87.6 Average 88.85 Standard Deviation 0.9

Finally, as a result of analyzing the proportion of NK cells, it was found that NK cells were present at a rate of 47.1% on the 6th day of culture. could know that More detailed results are shown in Table 4 below.

6 days N200515-01TS 67.9 N200515-02TS 68.1 N200612-01TS 20.9 N200612-02TS 31.5 Average 47.1 Standard Deviation 24.5

Example 3. Analysis of proliferation culture results - cell number / viability / NK cell ratio / cancer cell killing rate

After performing activation culture according to the method of Example 2, proliferation culture was performed for 6 days, and cell number, cell viability, NK cell ratio and cancer cell killing rate were measured.

First, as a result of the cell number analysis, as shown in Table 5 below, about 3 billion cells were obtained on the 6th day of the proliferation culture, and the following results were obtained as a result of 6 days of activation culture and 7 days of proliferation culture for each donor.

13 days (6 days + 7 days) N200515-01TS 3.66E+09 N200515-02TS 2.34E+09 N200612-01TS 2.62E+09 N200612-02TS 3.16E+09 Average 2.95E+09 Standard Deviation 5.86E+08

Next, the results of analyzing the cell viability are as shown in Table 6 below, and on the 6th day of culture, a viability of 93.2% was shown.

13 days (6 days + 7 days) N200515-01TS 94.5 N200515-02TS 95.2 N200612-01TS 90.8 N200612-02TS 92.4 Average 93.2 Standard Deviation 2.0

Next, the proportion of NK cells was measured through FACS. As a result, as shown in Figure 1, NK cells expressing CD56 accounted for 94.42% of the total, CD3 (T cells) was 2.10%, CD19 (B cells) was 1.84%, and CD14 (monocytes) was 0.79%. indicated.

Finally, it was attempted to analyze the killing rate of the cultured NK cells against cancer cells. For this purpose, NK cells cultured for 6 days in activation culture and proliferation culture for 7 days were used, and cells (samples 1, 2, 3) prepared from different donors were treated in K562 thymoma cell line. As a result, as can be seen in FIG. 2 , it was found that NK cells exhibited an average cancer cell killing rate of 70%.

In addition, FIG. 3 shows a photomicrograph showing the activated NK cells during the culture process.

Through the above examples, it was confirmed that the NK cell manufacturing process according to the present invention dramatically increases the relative proportion of NK cells from 5 to 15% to 94.42%, and increases the killing ability against cancer cells by about 70%. , it was found that it is possible to manufacture high-purity and high-activity NK cell therapeutics.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (8)

Interleukin-2 (Interleukin-2), interleukin-12 (Interleukin-12) and anti-CD16 antibody as an active ingredient, comprising a composition for enhancing the proliferation and activity of natural killer cells (Natural killer cells). The method of claim 1, The composition is characterized in that it further comprises one or more selected from the group consisting of interleukin-15, interleukin-18, anti-CD3 antibody and anti-CD56 antibody. The method of claim 1, The composition, characterized in that it further comprises autologous serum. (a) isolating monocytes from the peripheral blood of mammals including humans; (b) putting the mononuclear cells and the culture solution into a culture flask coated with at least one selected from the group consisting of anti-CD3 antibody, anti-CD16 antibody and anti-CD56 antibody, and then primary culturing for 5 to 7 days; and (c) recovering the cultured cells and a second culture for 5 to 7 days with a culture medium, comprising the step of, natural killer cells (Natural killer cells) manufacturing method, The culture medium is one or more selected from the group consisting of interleukin-2, interleukin-12, interleukin-15 and interleukin-18, and autologous A method of manufacturing, characterized in that it contains serum. 5. The method of claim 4, The autologous serum is characterized in that 5 to 15% is added, the manufacturing method. Claim 4 prepared by the method, natural killer cells (Natural killer cells). A cell therapy product for cancer treatment, comprising the natural killer cells of claim 6 as an active ingredient. A cell therapy agent for the treatment of infectious diseases, comprising the natural killer cells of claim 6 as an active ingredient.

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