Methods for preparing high purity natural killer cells with high efficiency, and use thereof

Abstract

To provide methods for preparing high purity natural killer cells with high efficiency, and uses thereof.SOLUTION: The disclosure provides a kit for NK cell activation and proliferation, the kit comprising: i) a culture flask coated with anti-CD3 antibody, anti-CD16 antibody and anti-CD56 antibody for NK cell activation; and ii) a culture medium comprising IL-2, IL-12, IL-15, IL-18 and an autologous serum to activate and proliferate NK cells. Also provided is a method for producing natural killer cells with the kit comprising the steps of: (a) isolating monocytes from the peripheral blood of a mammal including human; (b) culturing the monocytes with a culture medium comprising L-2, IL-12, IL-15, IL-18 and an autologous serum in the culture flask coated with anti-CD3 antibody, anti-CD16 antibody and anti-CD56 antibody for NK cell activation for from five to seven days; and (c) harvesting the cultured cells and proliferating the cells by conducting secondary culture of the cells with the culture medium for from five to seven days.SELECTED DRAWING: Figure 2

Description

Detailed Description of the Invention

[0001] [Technical field] The present invention relates to a method for producing highly pure and efficient Natural Killer cells (NK cells), and more particularly to a composition for enhancing the proliferation and activity of NK cells, and a method for producing the NK cells with enhanced proliferation and activity.

[0002] [Background technology] In cancer patients, the course of treatment using anticancer drugs has the side effect of reducing the patient's immune cells and weakening the immune function. Therefore, a treatment technique that transplants immune cells that have been proliferated and activated outside the body into the patient's body to activate the number and function of reduced immune cells can be expected to have a synergistic effect when used in parallel with conventional anticancer treatments, and can prevent cancer recurrence by having an effect of removing residual cancer and microresidual cancer after conventional anticancer treatment.

[0003] Natural killer cells (NK cells) used in immune cell therapy are cells that morphologically have large granules in the cytoplasm and account for about 5-15% of lymphocytes in the blood. NK cells are cytotoxic lymphocytes that differentiate from common lymphoid progenitors like T cells and B cells and play a role in eliminating tumor cells and virus-infected cells. However, unlike existing B cells, T cells, and dendritic cells (DC cells), NK cells can recognize cancer cells themselves without an activation process such as antigen presentation, and can directly kill cancer cells using enzymes such as granzymes and perforin. It has been reported that defects in the differentiation and activity of such NK cells are associated with various cancers such as breast cancer, melanoma cancer, and lung cancer, and the excellent killing ability of NK cells is applied to new immune cell therapies for the treatment of solid cancers and infectious diseases, and for preventing rejection of bone marrow and organ transplants.

[0004] However, despite the possibility of NK cells as a therapeutic agent for various diseases as mentioned above, the number of cells present in the body is not large, and in order to effectively use NK cells in cell therapy, it is most important to secure a large number of NK cells. However, the number of NK cells is small even in normal subjects, and in cancer patients, the number, differentiation, and function of NK cells are particularly reduced, making it difficult to secure a sufficient number of cells for use in therapeutic agents, and it is not easy to mass-multiply and culture them in vitro.

[0005] In this respect, various studies have been conducted on culture techniques for amplifying and activating NK cells to a practically useful level. For example, it has been reported that the conventional NK cell culture technique using IL-2 or other cytokines and chemicals was unable to dramatically increase the number of NK cells compared to the early days, and it has been reported that the proliferation of NK cells was effectively improved using irradiated cancer cells, MICA, 4-1BBL, and K562 cells transformed to express IL-15 as a culture technique using support cells (Tissue Antigens, 76(6):p467-475, 2010). However, these techniques all use cancer cells and have limitations in that they use methods that are not suitable for ensuring important safety in clinical applications, such as the possibility that the cancer cells may be mixed into the cell therapy drug formulation.

[0006] Therefore, there is a strong demand for a method for obtaining highly pure and highly efficient NK cells that can overcome the above conventional problems and can secure a large amount of NK cells that have sufficient cancer cell killing ability.

[0007] Summary of the Invention [Problem to be solved by the invention] The present inventors have conducted extensive research to establish an optimized NK cell production technique that overcomes the above-mentioned conventional problems and can obtain highly pure and highly efficient NK cells. As a result, they have developed a method for producing NK cells in which the proliferation and activity are significantly enhanced by only adding CD antibody and cytokines, without using supporting cells such as cancer cells, including a process of dividing culture for the purpose of activating and proliferating NK cells in blood, thereby completing the present invention.

[0008] Accordingly, an object of the present invention is to provide a composition for enhancing the proliferation and activity of natural killer cells (NK cells).

[0009] Another object of the present invention is to provide a method for producing natural killer cells (NK cells) with enhanced proliferation and activity.

[0010] However, the technical problems that the present invention aims to solve are not limited to the problems described above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

[0011] [Means for solving the problems] In order to achieve the above-mentioned object of the present invention, the present invention provides a composition for promoting the proliferation and activity of natural killer cells, comprising interleukin-2, interleukin-12 and anti-CD16 antibody as active ingredients.

[0012] In one embodiment of the present invention, the composition further comprises at least one selected from the group consisting of interleukin-15, interleukin-18, anti-CD3 antibody, and anti-CD56 antibody.

[0013] In another embodiment of the present invention, the composition further comprises autologous serum.

[0014] The present invention also provides a method for producing naturally occurring cytotoxic cells, comprising: (a) isolating monocytes from peripheral blood of a mammal, including a human; (b) placing the monocytes and a culture medium in a culture flask coated with one or more antibodies selected from the group consisting of anti-CD3 antibody, anti-CD16 antibody, and anti-CD56 antibody, and then performing a primary culture for 5 to 7 days; and (c) recovering the cultured cells and performing a secondary culture for 5 to 7 days together with the culture medium; wherein the culture medium contains one or more antibodies selected from the group consisting of interleukin-2, interleukin-12, interleukin-15, and interleukin-18, and autologous serum.

[0015] In one embodiment of the present invention, the autologous serum is added to 5 to 15%.

[0016] The present invention also provides a naturally killed cell produced by the method.

[0017] The present invention also provides a cell therapy agent for treating cancer, comprising the naturally occurring killing cells as an active ingredient.

[0018] The present invention also provides a cell therapeutic agent for treating infectious diseases, comprising the naturally occurring killing cells as an active ingredient.

[0019] The present invention also provides a method for preventing or treating cancer or an infectious disease, comprising administering the natural killing cells to an individual in need thereof.

[0020] The present invention also provides a use of said naturally killed cells for the manufacture of a medicament for the prevention or treatment of cancer or an infectious disease.

[0021] [Effects of the Invention] The NK cell production technique according to the present invention can obtain highly pure and highly efficient NK cells by significantly improving the proliferation and activity of NK cells, and overcomes the limitations of the conventional technique of using cancer cells as supporting cells. Therefore, the NK cells produced by the method according to the present invention can 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 cancer cell killing ability.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results of FACS analysis carried out to examine the proportion of NK cells in the culture obtained after the primary activation culture and the secondary expansion culture in the process for producing NK cells according to the present invention.

[0023] FIG. 2 shows the results of analyzing the cancer cell killing rate of NK cells obtained after primary activation culture and secondary proliferation culture in the process for producing NK cells according to the present invention.

[0024] FIG. 3 shows microscopic images of activated NK cells during the primary activation culture and secondary proliferation culture in the process for producing NK cells according to the present invention.

[0025] [Mode for carrying out the invention] The present invention will be described in detail below.

[0026] The present inventors have conducted extensive research to overcome the limitations of the conventional NK cell production techniques and establish an optimized NK cell production method capable of obtaining highly pure and highly efficient NK cells. As a result, they have developed a method capable of producing NK cells with enhanced proliferation and activity by only adding CD antibody and cytokines, thereby completing the present invention.

[0027] Accordingly, the present invention provides a composition for enhancing the proliferation and activity of natural killer cells, comprising interleukin-2, interleukin-12 and anti-CD16 antibody as active ingredients.

[0028] As used herein, the term "cell proliferation" refers to the proliferation or differentiation of cells in a culture as cells undergo a series of cell division steps. In the present invention, "proliferation of natural killer cells (NK cells)" refers to the proliferation of NK cells in a culture as NK cells undergo a series of cell division steps, or the proliferation of cells by differentiation of immature blood cells into NK cells.

[0029] The term "enhancing the activity of natural killer cells (NK cells)" as used herein includes both the transformation of immature NK cells into mature NK cells, or the activation of inactive NK cells, thereby increasing the number of cells capable of killing tumor cells or virus-infected cells in culture, or enhancing the cell-killing efficiency of individual NK cells.

[0030] In the present invention, the NK cells used as the source for the proliferation and activity enhancement can be commercially purchased or obtained from humans or animals, and preferably are provided from a human in need of NK cell therapy.

[0031] Furthermore, the NK cells are isolated from any tissue source in the body, but are preferably contained in blood collected from the body, more preferably from whole blood, umbilical cord blood, bone marrow or peripheral blood, and most preferably from peripheral blood.

[0032] In 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.

[0033] The cytokines used in the present invention are of the interleukin type, which is a general term for proteinaceous biologically active substances produced by immune cells such as lymphocytes, monocytes, and macrophages. In the present invention, the cytokines are selected from the group consisting of interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), and interleukin-18 (IL-18), and in particular, IL-2 and IL-12 can be used, and preferably, any of IL-2, IL-12, IL-15, and IL-18 can be used.

[0034] The "anti-CD antibody" used in the present invention is selected from the group consisting of anti-CD16 antibody, anti-CD3 antibody, and anti-CD56 antibody. In particular, anti-CD16 antibody can be used. Preferably, any of the anti-CD16 antibody, anti-CD3 antibody, and anti-CD56 antibody can be used.

[0035] The composition for enhancing the proliferation and activity of NK cells of the present invention may further contain, in addition to the interleukin and anti-CD antibody, other essential components, including autologous serum, or other carriers or auxiliary substances for enhancing the proliferation and activity of NK cells.

[0036] In another aspect of the present invention, the present invention provides a method for producing naturally occurring cytotoxic cells, comprising: (a) isolating monocytes from peripheral blood of a mammal, including a human; (b) placing the monocytes and a culture medium in a culture flask coated with one or more antibodies selected from the group consisting of anti-CD3 antibody, anti-CD16 antibody, and anti-CD56 antibody, and then performing a primary culture for 5 to 7 days; and (c) recovering the cultured cells and performing a secondary culture for 5 to 7 days together with the culture medium; wherein the culture medium contains one or more antibodies selected from the group consisting of interleukin-2, interleukin-12, interleukin-15, and interleukin-18, and autologous serum.

[0037] In the step (a) of the present invention, the peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood of a mammal, preferably a human, and mainly include immune cells such as B cells, T cells, and natural killer cells, and granulocytes such as basophils, eosinophils, and neutrophils. The PBMCs may be prepared from peripheral blood collected from a living body by a conventional method. For example, in the present invention, the PBMCs are isolated from peripheral blood by a density gradient centrifugation method using Ficoll.

[0038] In addition, the peripheral blood mononuclear cells are obtained from an individual in need of treatment, i.e., autologous peripheral blood mononuclear cells. When the peripheral blood mononuclear cells are autologous, there is an advantage that even if some T cells are present in the expanded NK cell population, there is no need to remove T cells because all cells are derived from the patient himself / herself.

[0039] The step (b) of the present invention is a culturing step for activating NK cells within peripheral blood mononuclear cells.

[0040] In the step (b) of the present invention, the method of coating the anti-CD antibody on the culture flask is carried out by adding a coating solution prepared by mixing DPBS with the anti-CD3 antibody, anti-CD16 antibody and anti-CD56 antibody to the culture flask, dispersing the antibodies uniformly, and then leaving the flask at 37°C for 2 to 6 hours, preferably 4 hours, or in a refrigerated condition for 16 to 24 hours, preferably 18 hours, and then washing the flask to remove the coating solution.

[0041] The mononuclear cells isolated in step (a) and the culture medium are placed in the coated culture flask and then cultured for 5 to 7 days, preferably 6 days, but the number of culture days can be appropriately set within the above range in order to obtain the desired NK cells by those skilled in the art. The number of cells to be inoculated is 1×10 based on a T75 flask. 6 It is desirable to inoculate 10 cells or more cells.

[0042] In a specific embodiment of the present invention, as a result of the primary culture in step (b), an increase in cell number and a high cell viability of at least 87.6% were confirmed, and the proportion of NK cells increased from an average of 13.9% to 47.1%, and it was confirmed that the proportion of NK cells increased by an average of 15% when the activation culture period was increased by 1 day.

[0043] The step (c) of the present invention is an expansion culture step for significantly increasing the number of NK cells.

[0044] The step (c) of the present invention is carried out by recovering the cells cultured in the step (b) and culturing them for 5 to 7 days, preferably 6 days, in a culture medium having the same composition as in the step (b). However, the culture period may be appropriately set within the above range in order to obtain NK cells as desired by those skilled in the art.

[0045] In a specific embodiment of the present invention, as a result of the secondary culture in step (c), about 3×10 9 Among the individual cells, NK cells accounted for an extremely high proportion of 94.42%, and showed a high survival rate of 93.2%. They also demonstrated an average cancer cell killing rate of 70% against K562 cells, a thymoma cell line.

[0046] In the steps (b) and (c), the culture conditions are the usual cell culture conditions, i.e., about 37° C., CO 2 The culture vessel is an incubator. In the present invention, a culture flask, more preferably a T75 flask, is used as the culture vessel, but a person skilled in the art can appropriately select and apply commercially available culture dishes, flasks, plates, multi-well plates, and culture bags.

[0047] In the present invention, the basic medium added to the culture solution for producing NK cells may be a medium for animal cell culture commonly used in the art, such as RPMI1640 medium, CellGro medium, AIM-V medium, and XVIVO 20. In the present invention, RPMI1640 medium is used, but the type of medium may be appropriately selected and applied by those skilled in the art. In addition, the culture medium may further contain other components necessary for the proliferation and culture of NK cells, in addition to the additives described above, as necessary.

[0048] In the present invention, the autologous serum further added to the culture medium is added at 5 to 15%, preferably 7 to 12 v / v%, more preferably 9 to 11 v / v%, and most preferably 10 v / v%.

[0049] The present invention specifically demonstrated through examples that the NK cell production method can provide NK cells with high purity and high efficiency compared to PBMCs.

[0050] In yet another aspect, the present invention provides a naturally killed cell produced by the above method.

[0051] The present invention also provides a cell therapy agent for treating cancer, comprising the naturally occurring killing cells as an active ingredient.

[0052] The cancer may include any type of cancer, including solid cancer and blood cancer. Unlike blood cancer, solid cancer means cancer formed in an organ as a mass, and includes cancer occurring in most organs. There is no particular limitation on the cancers treatable by using the NK cells according to the present invention, and they include gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinal blastoma, head and neck cancer, salivary gland cancer, lymphoma, etc.

[0053] The present invention also provides a cell therapeutic agent for treating infectious diseases, comprising the naturally occurring killing cells as an active ingredient.

[0054] The infectious disease is a disease caused by infection with a virus or a pathogen, and includes all diseases transmitted through the respiratory tract, blood, skin contact, and the like.

[0055] Non-limiting examples of such infectious diseases include Hepatitis B, Hepatitis C, human papilloma virus (HPV) infection, Cytomegalovirus infection, viral respiratory diseases, and influenza.

[0056] The term "treatment" refers to any action in which the administration of said natural killer cells reverses or beneficially alters the symptoms of cancer or infectious disease in an individual.

[0057] The cell therapeutic agent may be provided as a cell therapeutic agent containing the active ingredient alone or containing one or more pharma- ceutically acceptable carriers, excipients, or diluents.

[0058] Specifically, the carriers can be, for example, colloidal suspensions, powders, saline solutions, lipids, liposomes, microspheres or nanospherical particles, which can be complexed or associated with a delivery vehicle and delivered in vivo using delivery systems known to those skilled in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers or fatty acids.

[0059] When the cell therapy agent is formulated, it is prepared using a diluent or excipient such as a typical suspending agent, preservative, or filler. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, and emulsions. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. For local administration, the cell therapy agent may be combined with an organic substance such as a biopolymer, or an inorganic substance such as hydroxyapatite, specifically, a collagen matrix, a polylactic acid polymer or copolymer, a polyethylene glycol polymer or copolymer, and chemical derivatives thereof. When the cell therapy agent is prepared in a dosage form suitable for injection, the naturally killed cells are dissolved in a pharma- ceutical acceptable carrier or frozen in a solution in which they are dissolved. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, 19th ed., 1995]. The cell therapy agent can be prepared in a unit dose form or in a multi-volume container by formulating it with a pharma- ceutically acceptable carrier and / or excipient using a method that can be easily performed by a person skilled in the art.

[0060] The term "administration" refers to the introduction of a given substance into an individual by a suitable method, and by "individual" we mean any organism, such as rats, mice, livestock, including humans, that can carry cancer or infectious disease. Specific examples include mammals, including humans.

[0061] The cell therapeutic agent can be administered parenterally, and the injection method can be selected from topical or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intradural injection, transdermal injection, intranasal injection, intraintestinal injection, local injection, sublingual injection, intrarectal injection, and intrathoracic injection.

[0062] The cell therapy agent is administered in a pharma- tically effective amount. The term "pharma- tically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical arts. Based on an adult patient weighing 70 kg, for example, about 1,000 to 10,000 cells / dose, 1,000 to 100,000 cells / dose, 1,000 to 1,000,000 cells / dose, 1,000 to 10,000,000 cells / dose, 1,000 to 100,000,000 cells / dose, 1,000 to 1,000,000,000 cells / dose, 1,000 to 10,000,000,000 cells / dose, or 1,000 to 100,000,000,000 cells / dose may be administered once or several times a day at regular intervals, or may be administered several times at regular intervals.

[0063] The present invention also provides a method for preventing or treating cancer or an infectious disease, comprising administering the natural killing cells to an individual in need thereof.

[0064] The term "prophylaxis" refers to any act of preventing or delaying the onset of cancer or infectious disease in an individual by administration of said natural killing cells.

[0065] The present invention also provides a use of said naturally killed cells for the manufacture of a medicament for the prevention or treatment of cancer or an infectious disease.

[0066] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention.

[0067] [Example] Example 1. Method for producing highly pure and efficient NK cells In order to produce highly pure and efficient NK cells, the present inventors conducted an experiment according to the following procedure.

[0068] 1) Collection of autologous blood After blood was collected from the human body, it was placed in a sodium heparin tube and a plain tube to prepare for blood separation.

[0069] 2) Culture flask coating 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 distributed over the entire surface area. After that, the flask was left at 37°C for 4 hours or in a refrigerator for 18 hours, after which the coating solution was removed and 10 ml of DPBS was added to wash once.

[0070] 3) Serum preparation Of the blood samples, the blood in the plain tubes was centrifuged at 2000 rpm for 30 minutes at 4° C. After centrifugation, the supernatant was collected and filtered using a 0.2 μm membrane filter, and then stored in a refrigerator for use in culture.

[0071] 4) Immune cell separation The blood in the heparin tube was transferred to the BSC, then transferred to a 50ml tube to check the total volume, and the same amount of DPBS was added and mixed. The same amount of ficoll as the blood was transferred to a 50ml tube, and the mixture of DPBS and blood was added to the tube containing the ficoll to prepare it thoroughly. The prepared tube was then centrifuged at 2000 rpm for 20 minutes at 4°C, and after centrifugation, the buffy coat layer was collected and transferred to a new 50ml tube. DPBS in a volume twice that of the collected buffy coat was added, and centrifuged at 1200 rpm for 5 minutes at 4°C. After centrifugation, the supernatant was removed, the cells were thoroughly dissolved, and then 20 ml of DPBS in a volume twice that of the collected buffy coat was added and centrifuged at 1200 rpm for 5 minutes at 4°C. The supernatant was then removed, the cells were dissolved, and the culture medium was added to resuspend them, and the cell count was measured.

[0072] 5) Activation 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 culture medium was placed in a T75 flask and more than 1,000,000 immune cells were seeded. The cells were incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 6 days.

[0073] 6) Expansion and culture of immune cells After the activation culture for 5 to 7 days, the culture products were all collected in 50 ml tubes and centrifuged at 1200 rpm for 5 min at 4°C. The supernatant was then removed, the cells were thawed, and culture medium was added to resuspend them, and the cell count was measured. The cells were suspended in RPMI culture medium containing IL-2, IL-12, IL-15, and IL-18 to a total volume of 200 ml, and seeded in 1000 ml of polyvinyl culture medium. At this time, 4% autologous serum was added. The cells were incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 6 days.

[0074] 7) Addition of medium to immune cells After 3 to 5 days, when the culture medium turned yellow and colonies were formed, the cell number and viability were measured, and culture medium was added to RPMI culture medium supplemented with IL-2, IL-12, IL-15, and IL-18 until the total volume reached 1000 ml.

[0075] 8) Recovery and loading of immune cells The culture medium was transferred to four 250 ml tubes, and centrifuged at 1200 rpm for 10 minutes at 4°C. The supernatant was discarded, the cells were thoroughly dissolved, and the 100 ml The cells were suspended in normal saline and the cell count and viability were measured.

[0076] <Example 2. Analysis of Activation Culture Results-Cell Number / Viability / NK Cell Ratio> The present inventors prepared NK cells with enhanced proliferation and activity by the process of Example 1. To this end, blood was first collected from four donors, and the characteristics of PBMC in the blood were analyzed, and the results are shown in Table 1 below.

[0077] [Table 1]

[0078] Next, PBMCs from the blood of each donor were subjected to activation culture using the method described in Example 1 with an inoculation cell number of 2 x 10^7 / 50 ml and an inoculation cell density of 4 x 10^5 / ml, and after culture, the cell number, survival rate, and proportion of NK cells were analyzed.

[0079] First, as a result of cell count analysis, as can be seen from Table 2 below, an average of 10.0 x 10^7 cells were recovered on the 6th day of culture, and at least 8.23 ​​x 10^7 cells were obtained.

[0080] [Table 2]

[0081] Next, the cell viability was measured. As a result, as shown in Table 3 below, the average cell viability was 88.85% on the sixth day, indicating a viability of at least 87.6%.

[0082] [Table 3]

[0083] Finally, the proportion of NK cells was analyzed, and it was found that NK cells were present at a ratio of 47.1% on the sixth day of culture, and when the activation culture period was extended by one day, the proportion of NK cells increased by an average of 15%. More detailed results are shown in Table 4 below.

[0084] [Table 4]

[0085] Example 3. Analysis of proliferation culture results - cell number / survival rate / NK cell ratio / cancer cell killing rate After the activation culture was performed according to the method of Example 2, the proliferation culture was carried out for 6 days, and the cell number, cell survival rate, NK cell ratio, and cancer cell killing rate were measured.

[0086] First, as a result of cell count analysis, as shown in Table 5 below, about 3 billion cells were obtained on the 6th day of expansion culture. As a result of performing activation culture for 6 days and expansion culture for 7 days for each donor, the following results were obtained.

[0087] [Table 5]

[0088] The cell viability was then analyzed, and the results are shown in Table 6 below. On day 6 of culture, the cell viability was 93.2%.

[0089] [Table 6]

[0090] Next, the proportion of NK cells was measured by FACS. As a result, as shown in Figure 1, NK cells expressing CD56 accounted for 94.42% of the total, CD3 (T cells) for 2.10%, CD19 (B cells) for 1.84%, and CD14 (monocytes) for 0.79%.

[0091] Finally, the cancer cell killing rate of the cultured NK cells was analyzed. To this end, NK cells that had undergone 6 days of activation culture and 7 days of proliferation culture were used to treat K562 thymoma cell line with cells produced from different donors (samples 1, 2, and 3). As a result, as shown in Figure 2, it was found that the NK cells showed an average cancer cell killing rate of 70%.

[0092] FIG. 3 shows micrographs showing activated NK cells during the culture process. Through the above examples, it was confirmed that the NK cell preparation process according to the present invention dramatically increases the relative ratio of NK cells from 5-15% to 94.42% and increases the killing ability of cancer cells by about 70%, so it was found that it is possible to prepare a highly pure and highly active NK cell therapeutic agent.

[0093] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. [Brief description of the drawings]

[0094] [Figure 1] 1 shows the results of FACS analysis carried out to examine the proportion of NK cells in the culture obtained after the primary activation culture and the secondary proliferation culture in the process for producing NK cells according to the present invention. [Diagram 2] 1 shows the results of analyzing the cancer cell killing rate of NK cells obtained after primary activation culture and secondary proliferation culture in the process for producing NK cells according to the present invention. [Diagram 3] 1 shows microscopic images of activated NK cells during the primary activation culture and secondary proliferation culture in the process for producing NK cells according to the present invention.

Claims

1. A composition for promoting the proliferation and activity of natural killer cells, comprising interleukin-2, interleukin-12 and an anti-CD16 antibody as active ingredients.

2. The composition according to claim 1, further comprising at least one selected from the group consisting of interleukin-15, interleukin-18, anti-CD3 antibody, and anti-CD56 antibody.

3. The composition of claim 1 , further comprising autologous serum.

4. (a) isolating mononuclear cells from peripheral blood of a mammal, including a human; (b) placing the mononuclear cells and a culture medium in a culture flask coated with at least one antibody selected from the group consisting of an anti-CD3 antibody, an anti-CD16 antibody, and an anti-CD56 antibody, and then performing a primary culture for 5 to 7 days; (c) recovering the cultured cells and culturing them secondary with a culture medium for 5 to 7 days, The culture medium contains at least one selected from the group consisting of interleukin-2, interleukin-12, interleukin-15 and interleukin-18, and autologous serum.

5. The method according to claim 4, wherein the autologous serum is added to a concentration of 5 to 15%.

6. A naturally killed cell produced by the method of claim 4.

7. A cell therapy agent for cancer treatment, comprising the natural killing cells according to claim 6 as an active ingredient.

8. A cell therapy agent for treating infectious diseases, comprising the natural killer cells according to claim 6 as an active ingredient.

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