Genetically engineered cell lines for the activation and expansion of NK cells and uses thereof

Abstract

The present invention relates to genetically engineered cells for activating NK cells. According to one embodiment, the genetically engineered cells for activating NK cells can synergistically induce the proliferation and activation of NK cells from a sample, and can be usefully used in a method for proliferating NK cells, a method for measuring the proliferated NK cells, NK cell activity, or a method for diagnosing a disease associated with NK cell activity.

Description

[Technical field]

[0001] This application claims priority to Korean Patent Application No. 10-2020-0069854, filed on June 9, 2020, and Korean Patent Application No. 10-2021-0046106, filed on April 8, 2021, the entire disclosures of which are incorporated herein by reference.

[0002] The present invention relates to a genetically engineered cell line for activating and expanding NK cells, and uses thereof, and more particularly to a method for expanding NK cells using the cell line, or a method for measuring the activity of NK cells using the cell line. [Background technology]

[0003] NK cells (natural killer cells) are a type of cytotoxic lymphocyte that plays an important role in innate immunity. NK cells react to virus-infected cells and cancer cells, and their reactions are influenced by signals that are sent by various activating and inhibitory receptors of NK cells in response to their respective ligands on target cells. In general, if the activating signal is stronger than the inhibitory signal, the NK cells can attack the target cells, but if the inhibitory signal is even stronger, they will not attack. Therefore, normal cells are not attacked by NK cells because they have the inhibitory receptor ligand (MHC: major histocompatibility complex) of the NK cells and send inhibitory signals.

[0004] Some cancer cells have abnormalities in MHC on the cell surface, resulting in a decrease in the number of MHC. In such cases, there is no inhibitory signal for NK cells, and the NK cells attack the target cells. Perforin is secreted, which creates holes in the cell membrane of infected cells or cancer cells, and granzymes are released into the holes, resulting in cytotoxicity that kills the cells. In patients with B-cell lymphoma and many other cancers, defects have been found in the number and anticancer activity of NK cells, and it is known that functional abnormalities of NK cells are closely related to the occurrence of such cancers.

[0005] Therefore, therapies for cancer patients that utilize the anticancer ability of such NK cells are gaining momentum, but it is important to develop an expansion method to secure a large number of NK cells with such capabilities. In addition, it is known that the decreased function or abnormality of NK cells is related to cancer and various diseases, so there is a demand for the development of a method to measure the activity of NK cells.

[0006] Previously reported methods for expanding NK cells have involved using a variety of expensive cytokines at high concentrations, or using peripheral blood mononuclear cells or cancer cell lines together. However, these methods are not only costly, but also have a low expansion rate. In addition, many studies have shown that other lymphocytes, such as T cells, are expanded in addition to NK cells, making them unsuitable for NK cell-based immune cell therapy. Therefore, the development of an efficient method for selectively expanding only NK cells has become a major issue, and although research into this has been conducted (Korean Patent Registered 10-1525199), the current situation is still insufficient.

[0007] Meanwhile, the cytotoxicity test, which is a method for testing NK cell activity, is a test that examines the ability of NK cells to directly kill target cancer cells (K562 cell line) and is currently used in clinical laboratories. Recently, a cytotoxicity test using whole blood, which does not require a cell separation process, has been introduced instead of peripheral blood mononuclear cells, which require a complicated cell separation process. However, when using whole blood, various types of cytokines are required, and there is a problem of increased testing costs, which needs to be overcome. Summary of the Invention [Problem to be solved by the invention]

[0008] One embodiment provides cell lines, or feeder cells, genetically engineered to express membrane bound interleukin-18 (mbIL-18) and membrane bound interleukin-21 (mbIL-21).

[0009] Another embodiment provides a method of expanding NK cells comprising obtaining a blood sample containing a mixed population of NK cells and contacting at least a portion of the population of NK cells with genetically engineered cells to activate the NK cells, the genetically engineered cells being genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21).

[0010] Yet another embodiment provides a method of testing NK cell activity comprising obtaining a blood sample comprising a mixed population of NK cells, contacting at least a portion of the mixed population of NK cells with genetically engineered cells to activate the NK cells, the genetically engineered cells being genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21), and analyzing the degree of activation of the activated NK cells.

[0011] Yet another embodiment provides a method for diagnosing or providing diagnostic information for an NK cell associated disorder, comprising the steps of obtaining a blood sample comprising a mixed population of NK cells; contacting at least a portion of the mixed population of NK cells with genetically engineered cells to activate the NK cells, the genetically engineered cells being genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21); and analyzing the degree of activation of the activated NK cells. [Means for solving the problem]

[0012] One embodiment provides cell lines genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21).

[0013] The cell line is also used as a culture support cell that selectively expands only NK cells. Generally, the K562 cell line, which is a representative trophic support cell used for NK cell proliferation, is used only for the proliferation of NK cells, so the K562 cell line itself must not proliferate. Therefore, before culturing with NK cells, the K562 cell line is pretreated by irradiating it with strong radiation (e.g., 50 to 100 Gy) so that the K562 cell line itself does not proliferate at all and only helps the proliferation of the NK cells. Meanwhile, for efficient proliferation of the NK cells, most cytokines such as IL-2 and IL-15 must be continuously exposed to the NK cells. Therefore, when the cancer cell line-based trophic support cell is genetically engineered to express cytokines such as IL-2 and / or IL-15, the cancer cell line itself that has been pretreated by irradiation cannot survive for a long period of time during the culture process, and the selective proliferation efficiency of the NK cells is reduced. In one embodiment, it was confirmed that when NK cells were exposed to IL-18 or IL-21 for a short period in the presence of K562, no cell expansion effect was observed until day 21, but when exposed to IL-18 and IL-21 simultaneously, the cell expansion effect was observed after day 28. Therefore, it can be seen that IL-18 and IL-21 are effective in expanding NK cells despite only one exposure on the start day of culture and for a short period of exposure. That is, according to one embodiment, a culture support cell line genetically engineered to express mbIL-18 and mbIL-21 can selectively expand only NK cells without decreasing the NK cell expansion efficiency despite short-term expression of IL-18 and IL-21.

[0014] The term "feeder cells" as used herein may refer to cells that are not capable of dividing and proliferating when irradiated with radiation, but have metabolic activity and produce various metabolic substances, thereby helping the proliferation of target NK cells. The feeder cells that may be used herein include animal cell lines into which genes have been introduced, such as human chronic myeloid leukemia cell lines (e.g., K562 cells), RPMI 8866, EBV_LCL, 721.221, HFWT, and NK-92.

[0015] As used herein, the terms "genetic engineering" or "genetically engineered" refer to the act of introducing one or more genetic modifications into a cell, or to the cell produced thereby.

[0016] In particular, the genetically engineered cells may also comprise an exogenous gene coding for said gene. The term "exogenous" means that the referenced molecule or the referenced activity has been introduced into the host cell. The molecule may be introduced into the host genetic material, for example by insertion of an encoding nucleic acid into the host chromosome, or as non-chromosomal genetic material, such as a plasmid. In relation to the expression of the encoding nucleic acid, the term "exogenous" indicates that the encoding nucleic acid has been introduced into an individual in an expressible form. In relation to a biosynthetic activity, the term "exogenous" refers to an activity that has been introduced into the host mother cell. The source may also be, for example, a homologous or heterologous encoding nucleic acid that expresses the referenced activity after being introduced into the host mother cell. Hence, the term "endogenous" refers to the referenced molecule or activity that is present in the host cell. Similarly, with reference to expression of an encoding nucleic acid, the term "endogenous" refers to expression of an encoding nucleic acid contained within an individual. The term "heterologous" refers to a molecule or activity from another source other than the referenced species, and the term "homologous" refers to a molecule or activity from the host mother cell. Thus, exogenous expression of an encoding nucleic acid can utilize either or both of a heterologous or homologous encoding nucleic acid.

[0017] Thus, the cells may contain nucleic acid encoding mbIL-18 and mbIL-21.More particularly, the cells may be transformed with a vector containing nucleic acid encoding mbIL-18 and mbIL-21.

[0018] The term "vector" as used herein refers to a gene construct capable of expressing a target protein in a suitable host cell and including a regulatory element operably linked to allow a gene insert to be expressed. The vector according to one embodiment may include expression regulatory elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and / or an enhancer, and the promoter of the vector may be constitutive or inducible. The vector may also be an expression vector capable of stably expressing the fusion protein in a host cell. The expression vector may be a vector generally used in the art to express a foreign protein in a plant, animal, or microorganism. The recombinant vector may also be constructed by various methods known in the art. For example, the vector may include a selectable marker for selecting a host cell containing the vector, and may include a replication origin if the vector is replicable. The vector may also be self-replicating or introduced into a host or host DNA, and the vector may be selected from the group consisting of a plasmid, a lentivirus, an adenovirus, an adeno-associated virus, a retrovirus, a herpes simplex virus, and a vaccinia virus.

[0019] In the vector, the polynucleotide sequence encoding the fusion protein may be operably linked to a promoter. As used herein, the term "operably linked" refers to a functional association between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0020] As used herein, the term "membrane bound interleukin" refers to an interleukin bound to a cell membrane, and may be distinguished from interleukins secreted outside the cell. mbIL-18 may have sequence homology of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the nucleic acid sequence of SEQ ID NO: 1 or its amino acid sequence. mbIL-21 may have sequence homology of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more with the nucleic acid sequence of SEQ ID NO: 2 or its amino acid sequence.

[0021] Another embodiment provides a composition for NK cell culture comprising cells genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21).

[0022] The specific details of the genetically engineered cells are as described above.

[0023] In one embodiment, a composition for NK cell culture comprising cells genetically engineered to express mbIL-18 and mbIL-21 can induce the expansion and / or activation of NK cells (e.g., natural killer cells) and can be usefully used for the culture, isolation or proliferation of NK cells.

[0024] Yet another embodiment provides a method of expanding NK cells comprising obtaining a blood sample comprising a mixed population of NK cells and contacting at least a portion of the mixed population of NK cells with genetically engineered cells to activate the NK cells, the genetically engineered cells being genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21).

[0025] In one embodiment, the contacting step may also include co-culturing the genetically engineered cells with a mixed population of NK cells to stimulate, activate or expand a subpopulation of the NK cells.

[0026] As used herein, the term "stimulation of NK cells" can mean increasing the activity, e.g., cytotoxic activity, of natural killer cells, or generating, increasing, amplifying or proliferating activated natural killer cells in vitro or in vivo.

[0027] In one embodiment, non-limiting examples of the NK cells include macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils. Thus, in a particular embodiment, the NK cells are any one selected from the group consisting of macrophages, B lymphocytes, T lymphocytes (CD8+ CTL), mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils. In a particular embodiment, the NK cells are natural killer cells or T lymphocytes. The mixed NK cells may include one or more selected from the group consisting of macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils.

[0028] As used herein, the term "natural killer cells" or "NK cells" refers to cytotoxic lymphocytes that constitute a major component of the innate immune system, and are defined as large granular lymphocytes (LGLs), which constitute a third type of cells differentiated from B and T lymphocytes that are generated by common lymphoid progenitors (CLPs). The aforementioned "natural killer cells" or "NK cells" include natural killer cells without further transformation derived from any tissue source, and may include not only mature natural killer cells but also natural killer progenitor cells. The natural killer cells are activated by a response to interferon or macrophage-derived cytokines, and the natural killer cells contain two types of surface receptors that control the cytotoxic activity of the cells, also labeled "activating receptors" and "inhibitory receptors". The natural killer cells can also be generated from hematopoietic cells, such as hematopoietic stem cells or progenitors, from any source, such as placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc.

[0029] In one embodiment, the natural killer cells are also activated natural killer cells. The activated natural killer cells may refer to cells in which the cytotoxicity or natural immunomodulatory ability of natural killer cells is activated compared to parent cells, such as hematopoietic cells or natural killer progenitor cells.

[0030] In one embodiment, activated innate killer cells, or enriched populations of activated innate killer cells, may also be assessed by detecting one or more functionally related markers, such as CD16, CD57, CD69, CD94, CD161, CD158a, CD158b, NKp30, NKp44, NKp46, DNAM-1, 2B4, NKp46, CD94, KIR (e.g., KIR2DL1, KIR2DL2 / 3, KIR3DL1), and the NKG2 family of activating receptors (e.g., NKG2A, NKG2C, NKG2D).

[0031] In one embodiment, the co-culture may be performed in the presence of cytokines.

[0032] The term "cytokine" as used herein may refer to a protein (5-20 kDa) that plays a role in cell signaling. The cytokines are released by cells and affect the behavior of the cell that releases the cytokine and / or other cells. Non-limiting examples of cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, monokines, and colony stimulating factors. The cytokines are also produced by a wide range of cells, including, but not limited to, immune cells, such as macrophages, B lymphocytes, T lymphocytes, mast cells and monocytes, endothelial cells, fibroblasts, and stromal cells. The cytokines may be produced by one or more types of cells. The cytokines act through receptors and are particularly important in the immune system, regulating the balance between humoral and cell-based immune responses and regulating the maturation, growth, and responsiveness of cell populations. The cytokines herein may be naturally occurring cytokines or mutated versions of naturally occurring cytokines. "Naturally occurring" as used in this application is also referred to as wild type and includes allelic variants. Mutated versions of the naturally occurring cytokines, or "mutations", refer to specific mutations made to the naturally occurring sequence to alter the function, activity and / or specificity of the cytokine. In one embodiment, the mutations can improve the function, activity and / or specificity of the cytokine. In other embodiments, the mutations can decrease the function, activity and / or specificity of the cytokine. The mutations can also include the deletion or addition of one or more amino acid residues of the cytokine.

[0033] The cytokine may be any one selected from the group consisting of BMP (Bone Morphogenetic Protein) family, CCL (chemokine ligands) family, CMTM (CKLF-like MARVEL transmembrane domain containing member) family, CXCL (CXC motif ligand) family, GDF (Growth / Differentiation Factor) family, growth hormone, IFN (interferon) family, IL (interleukin) family, TNF (Tumor Necrosis Factors) family, GPI (glycophosphatidylinositol), SLUPR-1 (secreted Ly-6 / uPAR-related protein 1), SLUPR-2 (secreted Ly-6 / uPAR-related protein 2), and combinations thereof.

[0034] In one embodiment, the cytokine is an interleukin or a mutant thereof. Many interleukins are synthesized by monocytes, macrophages and endothelial cells as well as auxiliary CD4 T lymphocytes. The interleukins can promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Non-limiting examples of the interleukins include IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35 or IL36. Thus, in certain embodiments, the cytokine is an interleukin, including, but not limited to, wild-type and mutant forms of IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35 or IL36, or a mutant thereof.

[0035] In another embodiment, the cytokines added to the co-culture are also IL-18 and IL-21. More specifically, the co-culture can include adding IL-18 and IL-21 to the culture medium while culturing in the presence of IL-2 and IL-15. The IL-21 is also used at a concentration of 1 to 20 ng / ml, 1 to 15 ng / ml, 1 to 10 ng / ml, or 2 to 8 ng / ml. The IL-18 is also used at a concentration of 10 to 200 ng / ml, 10 to 180 ng / ml, 20 to 160 ng / ml, 20 to 120 ng / ml, 20 to 40 ng / ml, 20 to 80 ng / ml, 40 to 160 ng / ml, 40 to 120 ng / ml, 40 to 80 ng / ml, 80 to 160 ng / ml, or 80 to 120 ng / ml. In one embodiment, NK cell proliferation can be further synergistically increased by exposing cells to said IL-18 and said IL-21 during co-culture.

[0036] In one embodiment, the co-cultivation may be for between 2 days and 30 days.

[0037] In one embodiment, the genetically engineered cells may also be irradiated with between 50 Gy and 300 Gy.

[0038] In one embodiment, the sample is a biological sample from an individual, such as a mammal, including a human. The biological sample may be isolated from an individual and may be blood, whole blood, serum, plasma, lymph, urine, feces, tissue, cells, organs, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof. The biological sample may include PBMCs, purified NK cells, or primary resting cells (i.e., freshly isolated from blood).

[0039] Yet another embodiment provides a method of testing NK cell activity comprising obtaining a blood sample comprising a mixed population of NK cells, contacting at least a portion of the mixed population of NK cells with genetically engineered cells to activate the NK cells, the genetically engineered cells being genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21), and analyzing the degree of activation of the activated NK cells.

[0040] Yet another embodiment provides a method for diagnosing or providing information regarding an NK cell associated disease, comprising the steps of obtaining a blood sample comprising a mixed population of NK cells; contacting at least a portion of the mixed population of NK cells with genetically engineered cells to activate the NK cells, the genetically engineered cells being genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21); and analyzing the degree of activation of the activated NK cells.

[0041] In the method for testing the activity of NK cells of the present invention, the step of comparing the activation of the NK cells with that of normal NK cells specifically includes determining that the activation phenomenon exhibited by the normal NK cells in the experimental group is significantly higher, not exhibited, or exhibited at a significantly lower level when the normal NK cells are stimulated with the genetically engineered cells under the same conditions as those in the experimental group, as abnormal. By the above step, pathological signs of diseases associated with abnormal NK cells, viral infection, the presence of cancer cells, and specific cancers can be determined, and the prognosis of the diseases can be predicted. The "normal NK cells" refer to NK cells possessed by or derived from an individual without a disease, and the individual without a disease does not have at least any physical, genetic, or external condition known to affect NK cell activity.

[0042] In one embodiment, the method may further include a step of isolating the desired NK cells from the sample. If necessary, the step may be performed after stimulation while the cells are present together with other blood cells or lymphocytes, or the step of stimulating may be performed after the step to obtain a sample containing only NK cells as lymphocytes. The degree of purification of the isolated NK cells and the composition of the sample may be as varied as required for the experiment. The NK cells may be used as they are purified from the sample, if necessary, or may be used after proliferation to ensure conditions or cell amount suitable for the experiment.

[0043] However, since certain combinations of factors are specific to NK cells, when using a combination of factors specific to the NK cells in the method of the present invention, the step of isolating the NK cells is not necessarily required.

[0044] In one embodiment, the step of measuring the degree of activation may include one or more selected from the group consisting of degranulation activity, cytotoxicity activity, and measurement of cytokines secreted upon NK cell stimulation.

[0045] The degranulation activity may refer to, for example, the induction of lysis of target cells by secreting perforin or granzyme, and may be analyzed by FACS. Specifically, after stimulating PBMCs isolated from a whole blood sample or pure isolated NK cells, a method may be used in which CD107a expression, which is proportional to degranulation, is measured using a fluorochrome-conjugated antibody.

[0046] The cytotoxic activity can be measured, for example, by culturing the cells with a culture containing target cells capable of activating NK cells labeled with europium fluorescent dye, and then measuring the amount of fluorescent dye released through target cell lysis using a microplate reader.

[0047] In one embodiment, the cytokine may be selected from IFN-γ, TNF-α, TNF-β, MIP-1α, MIP-1β, PANTES, IL-8, and IL-10. The expression of immune activators in NK cells may be analyzed using FACS, intracellular cytokine staining, ELISA, or the like. Specifically, a method may be used in which the surface of NK cells is stained using a specific fluorochrome-conjugated antibody, the cells are permeabilized, and the cytokine is stained with another specific fluorochrome-conjugated immune activator (e.g., IFN-γ antibody) to measure the expression of immune activators in NK cells.

[0048] In one embodiment, the disease associated with NK cell activity is one that exhibits abnormal NK cell activity, such as a hypersensitive immune disease, an autoimmune disease, an immune rejection reaction, an immune deficiency disease, histiocytosis, cancer, type 2 diabetes, a parasitic infection disease, and a viral disease. The hypersensitive immune disease may be one or more selected from asthma and sinusitis, or the autoimmune disease may be one or more selected from lupus, multiple sclerosis, type 1 diabetes, and rheumatoid arthritis, or the histiocytosis may be one or more selected from hemophagocytic lymphohistiocytosis (HLH), XLP1, and XLP2. The hemophagocytic lymphohistiocytosis (HLH) may also include group 2 Langerhans cell histiocytosis, erythrophagocytic lymphohistiocytosis (familial, sporadic), infection-associated hemophagocytosis syndrome, virus-associated hemophagocytosis syndrome, histiocytosis with bulky lymphadenopathy, or reticulohistiocytosis. The term "cancer" includes tumors, blood cancers, or solid cancers, and includes those that impair the synergistic activity of NK cells in an individual or do not cause the synergistic activity of NK cells as target cells under certain conditions. In one embodiment, the cancer may be selected from the group consisting of lung cancer, liver cancer, esophageal cancer, gastric cancer, colon cancer, small intestine cancer, pancreatic cancer, melanoma, breast cancer, oral cancer, brain cancer, thyroid cancer, parathyroid cancer, kidney cancer, cervical cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, testicular cancer, blood cancer, lymphoma, skin cancer, psoriasis, and fibroadenoma. In one embodiment, the cancer may be pancreatic cancer or B cell lymphoma. In one embodiment, the viral disease may be hepatitis B. In one embodiment, the immune deficiency disease may be DiGeorge syndrome or Chediak-Higashi syndrome.

[0049] Regarding information on the NK cell activity-related disease, if no activation is detected in the NK cells of the experimental group compared to normal NK cells, the NK cells can be determined to be abnormal, or if NK cells that have lost activity against a specific receptor are abnormally active or not active against target cells, the target cells can be determined to be related to a specific disease.

[0050] Yet another embodiment provides NK cells produced by the method for expanding NK cells.

[0051] In still another aspect, there is provided a cell therapeutic agent comprising the immune cells or a cell population thereof as an active ingredient.

[0052] In yet another embodiment, there is provided a pharmaceutical composition for preventing or treating cancer or an infectious disease, comprising the immune cells or a population of such cells as an active ingredient.

[0053] Yet another aspect provides the use of said immune cells or cell populations thereof for the manufacture of a medicament.

[0054] Yet another aspect provides a method for treating a disease comprising administering to an individual said immune cells or population of cells thereof.

[0055] As used herein, the term "disease" may refer to a pathological condition, in particular cancer, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune disorders, degenerative diseases, cell death-related diseases, and graft rejection.

[0056] As used herein, the term "treatment" refers to or includes the alleviation, slowing down or prevention of a disease, disorder or condition, or one or more symptoms thereof, and "active ingredient" or "pharmaceutical effective amount" may refer to any amount of a composition utilized in the course of practicing the inventions provided herein sufficient to alleviate, slow down or prevent a disease, disorder or condition, or one or more symptoms thereof.

[0057] As used herein, the terms "administer," "introduce," and "implant" are used interchangeably and may refer to the placement of a composition according to an embodiment into an individual by a method or route that results in at least partial localization of the composition according to an embodiment at a desired site. Administration may also be by any suitable route that delivers at least a portion of the cells or cellular components of a composition according to an embodiment to a desired location within a living individual. Following administration to an individual, the survival period of the cells may be as short as a few hours, e.g., 24 hours to a few days, or as long as several years.

[0058] As used herein, the term "isolated cells", e.g., "isolated immune cells", refers to cells that have been substantially separated from the tissue from which they originate, e.g., hematopoietic cells.

[0059] The method of administration of the pharmaceutical composition according to one embodiment is not particularly limited, and may be administered parenterally, such as intravenously, subcutaneously, intraperitoneally, by inhalation or topical application, or may be administered orally, depending on the intended method. The dosage ranges depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. A daily dosage refers to an amount of a therapeutic agent according to one embodiment that is sufficient to treat a disease state alleviated by administration to an individual in need of treatment. The effective amount of a therapeutic agent varies depending on the specific compound, the disease state and its severity, and the individual in need of treatment, and can be generally determined by one of ordinary skill in the art. As a non-limiting example, the dosage of a composition according to one embodiment to a human body may also vary depending on the patient's age, weight, sex, health condition, administration form, and disease severity. When an adult patient weighing 70 kg is used as a standard, the dosage is, 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, 1,000 to 100,000,000 cells / dose, 1,000 to 1,000,000,000 cells / dose, or 1,000 to 10,000,000,000 cells / dose, and it can be administered once a day at regular intervals, or in divided doses several times a day, or it can be administered several times at regular intervals.

[0060] By "individual" is meant a subject in need of treatment for a disease, and more specifically means a mammal, such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow.

[0061] The pharmaceutical composition according to one embodiment may contain pharma- ceutically acceptable carriers and / or additives, such as sterile water, saline, conventional buffers (phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonicity agents, or preservatives. For local administration, it may contain organic materials such as biopolymers, inorganic materials such as hydroxyapatite, specifically, collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof, in combination. When the pharmaceutical composition according to one embodiment is formulated into a dosage form suitable for injection, the immune cells or the substance that increases their activity may be dissolved or frozen in a solution state in a pharma- ceutically acceptable carrier.

[0062] The pharmaceutical composition according to one embodiment may appropriately contain a suspending agent, a solubilizing agent, a stabilizing agent, an isotonicity adjusting agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjusting agent, a soothing agent, a buffer, a reducing agent, an antioxidant, etc., if necessary, depending on the administration method and dosage form. 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 pharmaceutical composition according to one embodiment may be prepared into a unit dose form or may be placed in a multi-capacity container by formulating the composition using a pharma-ceutically acceptable carrier and / or excipient by a method that can be easily carried out by a person skilled in the art to which the present invention belongs. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or in the form of a powder, granule, tablet or capsule. Effect of the Invention

[0063] According to one embodiment, the genetically engineered cells can increase the proliferation and activity of NK cells by at least two to several times compared to non-genetically engineered cells, and can also be used as a cell therapy agent by expanding NK cells. [Brief description of the drawings]

[0064] [Figure 1A] FIG. 1 is a schematic diagram showing the genes of lentiviruses producing mbIL-18 and mbIL-21 for producing cultured accessory cells according to one embodiment. [Figure 1B] 1 is a graph confirming the expression characteristics of mbIL-18 and mbIL-21 in cultured accessory cells according to one embodiment. [Figure 2A] 1 is a graph showing the fold expansion (left) and purity (right) of NK cells when cultured accessory cells according to one embodiment and NK cells were cultured in complete RPMI 1640 medium. [Figure 2B]1 is a graph showing the expansion fold (left) and purity (right) of NK cells when cultured auxiliary cells according to one embodiment and NK cells were cultured in DS medium. [Figure 2C] 1 is a graph comparing the fold expansion (left) and purity (right) of NK cells when cultured accessory cells according to one embodiment and NK cells were cultured in complete RPMI 1640 medium and DS medium. [Figure 3A] 1 is a graph comparing markers expressed in expanded NK cells after expanding the NK cells using an embodiment of the supplemental culture cells (blue) and K562 supplemental culture cells (red) in RPMI 1640 medium and DS medium. [Figure 3B] 1 is a graph comparing markers expressed in expanded NK cells after expanding the NK cells using an embodiment of the supplemental culture cells (blue) and K562 supplemental culture cells (red) in RPMI 1640 medium and DS medium. [Figure 4A] 1 is a graph confirming the cytotoxicity of NK cells from healthy donors (HD) expanded using cultured accessory cells according to one embodiment. [Figure 4B] 1 is a graph confirming the ADCC cytotoxicity of NK cells from healthy donors (HD) expanded using cultured accessory cells according to one embodiment. [Figure 5A] 1 is a graph measuring CD107a expression in NK cells stimulated with cultured accessory cells according to one embodiment. [Figure 5B] 1 is a graph measuring CD107a expression in NK cells stimulated with cultured accessory cells according to one embodiment. [Figure 5C] 1 is a graph showing the target cell lysis rate of NK cells stimulated with cultured accessory cells according to one embodiment. [Figure 5D] 1 is a graph showing the target cell lysis rate of NK cells stimulated with cultured accessory cells according to one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention. EXAMPLES

[0066] Example 1. Production of genetically engineered cultured accessory cells A cultured accessory cell line expressing membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21) was prepared.

[0067] Specifically, human-derived mbIL18 gene (SEQ ID NO: 1) and mbIL21 gene (SEQ ID NO: 2) were cloned into the lentivirus vector pCDH-CMV-RFP to prepare a recombinant lentivirus production vector (FIG. 1A). Then, for virus production, the prepared recombinant gene (pCDH-CMV-RFP-mbIL-1821) was transfected into 293FT cells together with a packaging vector using lipofectamin3000 (Invitrogen). After a certain period of time, the medium was replaced with a new one, and the cells were cultured for 48 hours, after which the medium containing the virus was collected. The collected medium was centrifuged at 500×g for 10 minutes, and only the pure medium containing the virus was separated using a 0.45 μm filter, and mbIL18-mbIL21 expressing lentivirus was produced. Then, 1 ml of mbIL18-mbIL21 expressing lentivirus was dissolved in 9 ml of medium containing K562 cells, and added together with polybrene (8 μg / ml), and the cells were cultured for 48 hours. Next, to select only infected cells, an ultra-high speed automatic flow cell separator was used to select only cells expressing red fluorescence, and the expression of mbIL18-mbIL21 was analyzed using a flow cytometer (FACS), producing a K562 cell line expressing mbIL18-mbIL21 (hereinafter referred to as "K562-mbIL18-mbIL21").

[0068] [Experimental Example] Experimental example 1. Cellular characteristics of genetically engineered cultured accessory cells To ascertain the mbIL-18 and mbIL-21 expression characteristics of the genetically engineered cultured accessory cells, according to one embodiment, mRNA and surface protein expression levels were examined.

[0069] Specifically, total RNA of the K562-mbIL18-mbIL21 cell line and the K562 cultured support cell line prepared in Example 1 was isolated using RNeasy Mini Kit (Qiagen, Venlo, Netherlands) and quantified using IMPLEN Nanophotometer P330 (IMPLEN, Munich, Germany). The isolated RNA was then converted to cDNA using QuantiTect Reverse Transcription Kit (Qiagen), and PCR was performed in a standard reaction volume of 20 μl using QuantiTect SYBR Green PCR Kit (Qiagen) and Rotor-GeneQ (Qiagen). At that time, the primers in Table 1 below were used, and all experiments were repeated three times.

[0070] [Table 1]

[0071] FIG. 1B is a graph confirming the expression profile of mbIL-18 and mbIL-21 in cultured accessory cells according to one embodiment.

[0072] As a result, as shown in Figure 1B, mbIL-18mRNA and mbIL-21mRNA were detected in the auxiliary cultured cells of Example 1, but mbIL-18mRNA and mbIL-21mRNA were not detected in the K562 auxiliary cultured cells. That is, it can be seen that the genetically engineered auxiliary cultured cells according to one embodiment have the expression characteristics of mbIL-18 and mbIL-21.

[0073] Experimental Example 2. Activation and expansion of NK cells using K562-mbIL18-mbIL21 cells In one embodiment, NK cell activation and expansion by the culture medium of genetically engineered accessory cells was confirmed.

[0074] 2-1. Complete RPMI 1640 medium Specifically, the K562-mbIL18-mbIL21 cell line prepared in Example 1 was cultured in a T-75 flask containing 25 ml of complete RPMI 1640 medium at 37°C in an incubator supplied with 5% CO2. Then, the cells were centrifuged at 400 x g for 3 minutes, and the cell pellet was resuspended in 5 ml of complete RPMI 1640 medium to harvest the feeder cells. Then, in order to prevent excessive growth of the feeder cells, the feeder cells were irradiated with gamma rays at 100 Gy using a Gammacell 3000 Elan irradiator, and used in subsequent experiments.

[0075] For peripheral blood mononuclear cells (PBMCs) isolation, healthy donor whole blood:PBS was diluted to a 1:2 ratio (10 ml whole blood:20 ml PBS) and overlaid on 15 ml Lymphoprep. Then, cells were harvested from the buffy coat layer by centrifugation at 1,200 × g for 25 min at room temperature without brake (acceleration 1, deceleration 0) and washed three times with PBS at 400 × g for 7 min. The isolated peripheral blood mononuclear cells (3 × 10 6 , and 0.5 × 10 K562-mbIL18-mbIL21 irradiated at 100 Gy 6 The cells were seeded onto a 24-well plate containing 1 ml of NK cell medium, and 1 ml of NK cell medium containing 20 U / ml IL-2 was added to make the total medium volume 2 ml / well and the final concentration of IL-2 10 U / ml. The cells were mixed by gentle pipetting and then incubated at 37°C in 5% CO 2 On the 7th day of culture, 100 U / ml IL-2 and 5 ng / ml IL-15 were added to the medium, and the medium was replaced with new medium every 2-3 days for 14 days. K562 cells were used as a control.

[0076] The expansion of NK cells was confirmed using FITC (fluorescein isothiocyanate)-conjugated mouse anti-human CD3 monoclonal antibody and PE-Cy5-conjugated mouse anti-human CD56 monoclonal antibody. The purity of NK cells when treated with K562 cells and IL-2 / IL-15 was used as the baseline, and the expansion fold of each was confirmed.

[0077] FIG. 2A is a graph showing the fold expansion (left) and purity (right) of NK cells when cultured accessory cells according to one embodiment and NK cells were cultured in complete RPMI 1640 medium.

[0078] As a result, as shown in FIG. 2A, in the case of K562 cultured auxiliary cells, it was observed that NK cells increased up to the 35th day after culture, but no further expansion occurred thereafter. In addition, in the case of the cultured auxiliary cells of Example 1, it was observed that the NK cell expansion fold increased up to the 42nd day. In addition, it was observed that the purity of the NK cells cultured using the cultured auxiliary cells of Example 1 was significantly higher than that of the NK cells cultured using K562 cultured auxiliary cells. In other words, the cultured auxiliary cells according to one embodiment not only significantly increase the activity of NK cells, but also enable long-term culture of NK cells, so that NK cells can be mass-multiplied and used as a cell therapy agent.

[0079] 2-2.DS (DMEM + supplements) medium The same procedure as in Experimental Example 2-1 was carried out, except that DMEM culture medium supplemented with 4.5 g / L glucose and 584 mg / L glutamine, DS media published by Miller group (JOURNAL OF HEMATOTHERAPY 4: 149-158 (1995)), was used.

[0080] FIG. 2B is a graph showing the expansion fold (left) and purity (right) of NK cells when cultured support cells according to one embodiment and NK cells were cultured in DS medium.

[0081] As a result, as shown in Figure 2B, it was observed that the NK cell expansion fold increased up to day 42 in both the supplemental culture cells of Example 1 and the cells of Comparative Example 1. However, it was observed that the NK cell expansion fold increased significantly by about 2 to 6 times or more in the supplemental culture cells of Example 1 compared to the cells of Comparative Example 1. It was also observed that the purity of the NK cells cultured using the supplemental culture cells of Example 1 was significantly higher than that of the NK cells cultured using the K562 supplemental culture cells.

[0082] Based on the above results, the NK cell proliferation efficiency in RPMI 1640 medium and DS medium was compared.

[0083] FIG. 2C is a graph comparing the fold expansion (left) and purity (right) of NK cells when cultured with cultured accessory cells according to one embodiment and NK cells in complete RPMI 1640 medium and DS medium.

[0084] As a result, as shown in Figure 2C, it was observed that the cells of Example 1 had an increased NK cell expansion fold and purity compared to K562 supplementary culture cells, regardless of the type of medium. However, when comparing the same cells cultured in different media, it was observed that the cells of Example 1 had a significantly higher NK cell expansion fold in DS medium compared to complete RPMI 1640 medium. In other words, the supplementary culture cells according to one embodiment can selectively expand NK cells depending on the type of medium, but can have medium-selective characteristics.

[0085] Experimental Example 3. Phenotypic characteristics of NK cells expanded by K562-mbIL18-mbIL21 cells The phenotypic characteristics of NK cells expanded by K562-mbIL18-mbIL2 cells were observed in RPMI 1640 medium and DS medium.

[0086] Specifically, for the isolation of peripheral blood mononuclear cells (PBMCs), healthy donor whole blood:PBS was diluted to a 1:2 ratio (10 ml whole blood:20 ml PBS) and overlaid on 15 ml Lymphoprep. Next, the cells were harvested from the buffy coat layer by centrifugation at 1,200 × g for 25 min at room temperature without brake (acceleration 1, deceleration 0), and washed three times with PBS at 400 × g for 7 min. Then, 2 × 10 expanded healthy donor-derived NK cells were isolated. 5 After washing with mFACS buffer (PBS containing 1% FBS), cells were treated with APC-Cyanine7-conjugated mouse anti-human CD3 membrane antibody and PE-Cyanine7-conjugated anti-human CD56 membrane antibody for 15 minutes. Then, cells were obtained and stained with different fluorescently conjugated anti-human CD16, CD67, CD69, NK2G2A, NK2G2C, NKG2D, DNAM-1, NKp30, NKp46, KIR2DL1, KIR2DL2 / 3, and KIR3DL1 membrane antibodies for 30 minutes. Then, cells were washed with FACS buffer, and data were acquired using FACSCalibur and analyzed with Kazuka.

[0087] 3A and 3B are graphs comparing markers expressed in expanded NK cells after expansion of the NK cells using an embodiment of the supplemental culture cells (blue) and K562 supplemental culture cells (red) in RPMI 1640 medium and DS medium.

[0088] As a result, as shown in Figures 3A and 3B, it was observed that surface markers such as CD16, CD67, CD69, NK2G2A, NK2G2C, NKG2D, DNAM-1, NKp30, NKp46, KIR2DL1, KIR2DL2 / 3 and KIR3DL1 were expressed in NK cells expanded using cultured helper cells according to one embodiment.

[0089] That is, it can be seen that the NK cells expanded by the cultured helper cells according to one embodiment have the complete functional characteristics of NK cells.

[0090] Experimental Example 4. Confirmation of cytotoxicity of NK cells expanded by K562-mbIL18-mbIL21 cells To validate the efficacy of NK cells expanded by cultured accessory cells according to one embodiment as antibody therapeutics, the cytotoxicity of NK cells against target cells on day 14 was measured over a 4-hour period by a CFSE-based assay.

[0091] Specifically, target cells were added to FACS buffer, stained with 0.5 μM CFSE for 10 min at 37 °C, and washed twice with complete medium (RPMI or DS). Then, 5 × 10 target cells were added to the FACS buffer and stained with 0.5 μM CFSE for 10 min at 37 °C. 4 The cells were placed in triplicate in a 96-well round-bottom plate and mixed with the healthy donor (HD)-derived NK cells of Experimental Example 3 at effector-to-target (E:T) ratios of 0.5:1, 1:1, and 2:1. The plate was centrifuged at 1,500 rpm for 3 minutes, and then incubated at 37°C, 5% CO 2 The cells were then cultured in an incubator for 4 h. The mixed cells were then transferred to FACS tubes, and 1 μl of 1 mg / mL propidium iodide (PI) (SigmaAldrich, St. Louis, MO, USA) was added to each tube. The cells were then acquired with a FACSCalibur and analyzed using Kaluza software. The percentage of dead target cells (CFSF positive and PI positive) was calculated after subtracting the percentage of spontaneously dead target cells.

[0092] FIG. 4A is a graph confirming the cytotoxicity of NK cells from healthy donors (HD) expanded using cultured accessory cells according to one embodiment.

[0093] FIG. 4B is a graph showing the ADCC cytotoxicity of NK cells from healthy donors (HD) expanded using cultured accessory cells according to one embodiment.

[0094] As a result, as shown in FIG. 4A, it was confirmed that the NK cells expanded using the supplemental culture cells of Example 1 exhibited the same cytotoxicity as the NK cells expanded using K562 supplemental culture cells. In addition, it was confirmed that the cytotoxicity of the NK cells cultured in DS medium was the same as that of the NK cells cultured in RPMI medium. In addition, as shown in FIG. 4B, it was confirmed that the NK cells expanded using the supplemental culture cells of Example 1 exhibited similar ADCC activity in the Raji cell mixture bound to rituximab as the NK cells expanded using K562 supplemental culture cells. In addition, it was confirmed that the ADCC of the NK cells cultured in DS medium was similar to that of the NK cells cultured in RPMI medium. That is, the NK cells expanded using the supplemental culture cells according to one embodiment can be used as an antibody therapeutic agent exhibiting cytotoxicity.

[0095] Experimental Example 5. Confirmation of the activity of NK cells stimulated by K562-mbIL18-mbIL21 cells 5-1. Confirmation of degranulation activity by CD107a expression In one embodiment, to confirm the cell activity of NK cells stimulated by genetically engineered cultured accessory cells in whole blood, the degree of CD107a expression on the NK cell surface, which is proportional to degranulation, was measured. Specifically, 100 μl of healthy donor (HD) whole blood in Experimental Example 3, K562 cultured accessory cells, or 2×10 K562-mbIL18-mbIL21 cultured accessory cell line prepared in Example 1 were used. 5 NK cells were cultured in FACS tubes with PE-conjugated anti-human CD107a. After 1 hour, Monensin and brefeldin A (BD Biosciences) were added and cultured for an additional 4 hours. NK cells were then stained with anti-human CD3 and human CD56 antibodies to measure CD107a expression. 5A and 5B are graphs measuring CD107a expression in NK cells stimulated with cultured accessory cells according to one embodiment. As a result, as shown in Figure 5A, it was observed that the NK cells stimulated with the cultured helper cells of Example 1 had increased CD107a expression in an IL-2 concentration-dependent manner. Specifically, as shown in Figure 5B, it was observed that the NK cells stimulated with the cultured helper cells of Example 1 had significantly higher CD107a expression at the same IL-2 concentration than the NK cells stimulated with the cultured helper cells of K562. That is, the NK cell activation using the cultured accessory cells according to one embodiment can be more useful as a method for diagnosing NK cell activity than the use of the K562 cultured accessory cells. 5-2. Confirmation of cytotoxicity by cell lysis To determine the cytotoxicity of NK cells stimulated by genetically engineered accessory cells in whole blood according to one embodiment, the cytotoxicity of target cells was measured for 16-20 hours by CFSE-based assay. Specifically, target cells (K562 accessory cells or accessory cells of Example 1) were added to FACS buffer, stained with 0.5 μM CFSE for 10 min at 37°C, and washed twice with complete medium (RPMI or DS). Then, 5×10 target cells were added to FACS buffer and stained with 0.5 μM CFSE for 10 min at 37°C, and washed twice with complete medium (RPMI or DS). 4 The cells were placed in triplicate in a FACS tube, and the complete medium supplemented with IL-2 and the whole blood of a healthy donor (HD) were mixed at a volume ratio of 200 μl, 100 μl, and 50 μl. The FACS tube was incubated at 37° C., 5% CO 2 The cells were cultured in an incubator for 16–20 h. The mixed cells were then transferred to FACS tubes, and 1 μl of 1 mg / mL propidium iodide (PI) (SigmaAldrich, St. Louis, MO, USA) was added to each tube. The cells were then acquired with a FACSCalibur and analyzed using Kaluza software. The percentage of dead target cells (CFSF positive and PI positive) was calculated after subtracting the percentage of spontaneously dead target cells. 5C and 5D are graphs showing the target cell lysis rate of NK cells stimulated with cultured accessory cells according to one embodiment. As a result, as shown in Figure 5C, it was observed that the NK cells stimulated with the cultured helper cells of Example 1 had an increased target cell lysis rate in an IL-2 concentration-dependent manner. Specifically, as shown in Figure 5D, it was observed that the NK cells stimulated with the cultured helper cells of Example 1 had a significantly higher target cell lysis rate at the same IL-2 concentration than the NK cells stimulated with the cultured helper cells of K562. That is, the NK cell activation using the cultured accessory cells according to one embodiment can be more useful as a method for diagnosing NK cell activity than the use of the K562 cultured accessory cells. The above description of the present invention is for illustrative purposes only, and those skilled in the art to which the present invention pertains 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, the above-described embodiments should be understood to be illustrative in all respects and not limiting.

Claims

1. 1. A support cell for the culture of NK cells, genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21), comprising: Here, the cultured support cells and NK cells are cultured in DS medium.

2. The auxiliary culture cell according to claim 1, wherein the auxiliary culture cell is selected from the group consisting of K562, RPMI 8866, EBV_LCL, 721.221, HFWT and NK-92 cells.

3. The auxiliary culture cell of claim 1, comprising membrane-bound interleukin-21 and a nucleic acid encoding membrane-bound interleukin-21.

4. A composition for NK cell culture, comprising the support cell according to claim 1 .

5. obtaining a blood sample from a non-human individual, the blood sample containing a population of NK cells; contacting at least a portion of the population of NK cells in culture with genetically engineered cells to activate the NK cells, the genetically engineered cells being cells genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21), Here, the cultured support cells and NK cells are cultured in DS medium.

6. 6. The method of claim 5, wherein the contacting step comprises co-culturing the genetically engineered cells with a population of NK cells to expand a subpopulation of the NK cells.

7. The method of claim 6 , wherein the co-culture is performed in the presence of cytokines.

8. The method of claim 7, wherein the cytokine is one or more selected from the group consisting of IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8 (CXCL8), IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL20, IL21, IL22, IL23, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35 and IL36.

9. The method of claim 8, wherein the cytokines are IL-18 and IL-21.

10. The method of claim 6, wherein the co-cultivation is carried out for between 2 days and 30 days.

11. The method of claim 5 , wherein the blood sample is a whole blood sample.

12. 6. The method of claim 5, wherein the genetically engineered cells are irradiated with 50 Gy to 300 Gy.

13. The method of claim 1, further comprising obtaining a blood sample from a non-human individual, the blood sample comprising a population of NK cells; contacting at least a portion of the population of NK cells with genetically engineered cells to activate the NK cells, the genetically engineered cells being genetically engineered to express membrane-bound interleukin-18 (mbIL-18) and membrane-bound interleukin-21 (mbIL-21); and analyzing the degree of activation of the activated NK cells, Here, the cultured support cells and NK cells are cultured in DS medium.

14. The method of claim 13, wherein the blood sample is a whole blood sample.

15. The method of claim 13, wherein the step of analyzing the degree of activation of the NK cells includes one or more selected from the group consisting of degranulation activity, cytotoxicity activity, and measurement of cytokines secreted by NK cell stimulation.

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