Feeder cell for activation and amplification of NK cells and use thereof

Abstract

The present invention relates to a feeder cell for the activation and amplification of NK cells and use thereof. The feeder cell for NK cells expresses at least one of mbCD16, mbIL-12, and mbIL-18, along with HLA-E, thereby stimulating NK cells to increase the activation and amplification of the NK cells, and these NK cells can be used as a cell therapeutic agent and a pharmaceutical composition for the prevention or treatment of cancer or infectious diseases.

Description

Cultured feeder cells for activating and amplifying NK cells and their uses The present invention relates to cultured auxiliary cells for activating and amplifying NK cells and their use. Natural killer (NK) cells are a type of cytotoxic lymphocyte that plays a crucial role in innate immunity. They primarily respond to virus-infected cells and cancer cells. This response is influenced by signals generated by interactions between various activating and inhibitory receptors on NK cells and ligands on target cells. Normal cells possess ligands (e.g., MHC) for inhibitory receptors on NK cells, transducing inhibitory signals and thus activating the defense mechanism against NK cells. Conversely, infected or abnormal cells possess ligands for activating receptors on NK cells, transducing activating signals. These activating signals can be amplified by stimuli such as cytokines, disrupting the balance with the inhibitory signals, initiating an NK cell attack. NK cells secrete perforin, which punctures the cell membranes of infected or cancer cells, and release granzymes, which activate apoptosis signals and induce cell death. NK cells, with their ability to directly recognize and destroy cancer cells, are currently undergoing various research and clinical trials as a potential anticancer treatment strategy. In particular, adaptive NK cells, which respond to stimuli such as tumors and infections and remember their functions, have recently begun to attract attention, in addition to the characteristics of conventional NK cells. Adaptive NK cells exhibit characteristics similar to memory cells, such as increased expression of the NKG2C activating receptor after HCMV infection and long-term survival. Research is underway to utilize these adaptive NK cells in anticancer treatments to complement the limitations of conventional NK cells. Although the clinical significance of adaptive NK cells is being emphasized, especially in studies demonstrating the amplification power of these cells (Int. J. Mol. Sci. 2022, 23, 9426), there is still a lack of research on the characteristics of adaptive NK cells, such as FcεRIγ reduction and increased ADCC efficacy. Therefore, to effectively use adaptive NK cells in anticancer treatment, it is necessary to develop a selective amplification technology that focuses on securing sufficient cell numbers of adaptive NK cells to maximize their adaptive characteristics. [Prior Art Literature] [Patent Document] Korean Patent Publication No. 10-1525199 U.S. Patent Publication No. 10,995,317 The purpose of the present invention is to provide cultured auxiliary cells for activating and amplifying NK cells. In addition, the present invention aims to provide a composition for activating and amplifying NK cells including the above culture auxiliary cells. In addition, the present invention aims to provide a method for activating and amplifying NK cells using the above culture auxiliary cells or composition. In addition, the present invention aims to provide a method for examining the activity of NK cells using the above culture auxiliary cells or composition. In addition, the present invention aims to provide a method for providing information for diagnosing NK cell-related diseases using the above culture auxiliary cells or composition. In addition, the present invention aims to provide NK cells manufactured by the method for activating and amplifying the NK cells. In addition, the present invention aims to provide a cell therapy agent containing the NK cell as an effective ingredient. In addition, the present invention aims to provide a pharmaceutical composition for preventing or treating cancer or infectious disease, which contains the NK cell as an active ingredient. In addition, one aspect of the present invention aims to provide a use of the NK cells. In addition, one aspect of the present invention aims to provide a method for treating cancer or infectious disease using the NK cells. Cultured feeder cells for NK cell activation and amplification One aspect of the present invention provides a culture feeder cell for activating and expanding NK cells expressing human leukocyte antigen E; and at least one selected from the group consisting of membrane bound anti-CD16 antibody, membrane bound interleukin-12, and membrane bound interleukin-18. The "feeder cell" used in the present invention refers to a cell that cannot undergo mitosis but has metabolic activity and thus produces various metabolites to help the proliferation of target cells, and is used by treating with mitomycin C or irradiating with radiation such as X-rays or γ-rays to inactivate division. The feeder cell in the present invention is for the activation and expansion of NK cells, and any animal-derived cell known in the art can be used without limitation. According to one specific example of the present invention, the culture auxiliary cells may be selected from the group consisting of K562, RPMI8866, ARH77, EBV_LCL, 721.221, and HFWT. In general, the K562 cell line, a representative nutrient-supporting cell line used for NK cell proliferation, is only used for the proliferation of NK cells, so the K562 cell line itself should 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, but only helps the proliferation of NK cells. Meanwhile, for the efficient proliferation of NK cells, most cytokines such as IL-2 and IL-15 must be continuously exposed to NK cells. Therefore, when cancer cell line-based nutrient-supporting cells are genetically engineered to express cytokines such as IL-2 and / or IL-15, there is a problem that the cancer cell line itself, which has been pretreated with irradiation, etc., cannot survive for a long time during the culture process, so the selective proliferation efficiency of NK cells is reduced. However, the culture auxiliary cells for activating and amplifying NK cells according to the present invention can selectively and more efficiently obtain NK cells by genetically engineering them to express human leukocyte antigens. According to one specific example of the present invention, the NK cell may be an adaptive NK cell. The "NK cell or natural killer cell" used in the present invention is a cytotoxic lymphocyte that constitutes a major component of the innate immune system, defined as a large granular lymphocyte (LGL) and constitutes a third cell differentiated from B and T lymphocytes that produce common lymphoid progenitors (CLP). In NK cells, CD16 (FcγRIII) is a specific surface protein, and CD56 is also a major surface protein. However, CD3 expressed in T cells is lacking. As used herein, the term "adaptive NK cell" refers to a specialized NK cell capable of forming immunological memory and participating in the adaptive immune system. In addition, the adaptive NK cell is a subgroup of cells that does not have antigen specificity, survives longer in the body among NK cells, and has stronger antibody-dependent cell-mediated cytotoxicity (ADCC). For example, the NK cell or adaptive NK cell may be, but is not limited to, an NKG2C+ NK cell, an FcεRγ- NK cell, or the like. As used herein, "human leukocyte antigen (HLA)" refers to a cell membrane glycoprotein molecule expressed on the surface of human nucleated cells, which presents antigens to T lymphocytes, inducing adaptive immunity against invading antigens, and also protecting normal cells from the killing action of NK cells. The HLA may be HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, etc. According to one specific example of the present invention, the human leukocyte antigen E or HLA-E may comprise a nucleic acid sequence of SEQ ID NO: 1. Specifically, the HLA-E may have a 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 to the nucleic acid sequence of SEQ ID NO: 1 or its amino acid sequence. The culture feeder cells for activating and amplifying NK cells according to the present invention are genetically engineered cells or cell lines that express at least one of membrane-bound anti-CD16 antibody, membrane-bound IL-12, and membrane-bound IL-18 together with HLA-E on the cell membrane through genetic engineering. Here, "genetic engineering" or "genetically engineered" means an act of introducing at least one genetic modification into a cell or a cell produced thereby. Specifically, the feeder cells may comprise an exogenous gene encoding at least one selected from the group consisting of HLA-E; and membrane-bound anti-CD16 antibody, membrane-bound IL-12, and membrane-bound IL-18. Here, "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 as an encoding nucleic acid, such as by insertion into the host chromosome, or as a non-chromosomal genetic material such as a plasmid. With respect to expression of the encoding nucleic acid, the term "exogenous" indicates that the encoding nucleic acid has been introduced into the organism in a form capable of being expressed. With respect to biosynthetic activity, the term "exogenous" indicates that the activity has been introduced into the host parent cell. The source may be, for example, a homologous or heterologous coding nucleic acid that expresses the activity mentioned after being introduced into a host cell. Conversely, "endogenous" refers to the molecule or activity mentioned being present in the host cell. Similarly, with respect to expression of a coding nucleic acid, the term "endogenous" refers to expression of the coding nucleic acid contained within the organism. The term "heterologous" refers to a molecule or activity from a source other than the mentioned species, and the term "homologous" refers to a molecule or activity from the host cell. Thus, exogenous expression of a coding nucleic acid may utilize either or both heterologous or homologous coding nucleic acids. According to one specific example of the present invention, the culture auxiliary cell may comprise a nucleic acid encoding at least one selected from the group consisting of HLA-E; and membrane-bound anti-CD16 antibody, membrane-bound IL-12, and membrane-bound IL-18. In addition, according to one specific example of the present invention, the culture auxiliary cell may be transformed with a vector containing a nucleic acid encoding at least one selected from the group consisting of HLA-E; and membrane-bound anti-CD16 antibody, membrane-bound IL-12, and membrane-bound IL-18. The above vector refers to a genetic construct that is capable of expressing a target protein in a suitable host cell and includes regulatory elements operably linked to express a gene insert. The vector 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. In addition, the vector may be an expression vector that can stably express the target protein or a fusion protein thereof in a host cell. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed by various methods known in the art. For example, the vector may include a selection marker for selecting a host cell containing the vector, and, if it is a replicable vector, may include a replication origin. Additionally, the vector may be self-replicating or may be introduced into the 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. Additionally, in the above vector, the polynucleotide sequence encoding the aforementioned target protein or fusion protein may be operably linked to a promoter. As used herein, "operably linked" means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence. In addition, the "membrane bound anti-CD16 antibody (mbCD16)" used in the present invention refers to an anti-CD16 antibody bound to a cell membrane, and may be distinguished from an anti-CD16 antibody secreted outside the cell. According to one specific example of the present invention, the membrane-bound anti-CD16 antibody may be at least one selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, and fragments thereof. The above anti-CD16 antibody may include any antibody or fragment thereof that has a structure and function that specifically binds to and reacts with an antigen, i.e., CD16. Here, "antibody" refers to a specific protein molecule directed against an antigenic site. In the present invention, it refers to an antibody that specifically binds to each protein, and includes monoclonal antibodies, polyclonal antibodies, antibodies with a full-length chain structure (full-length antibodies), and recombinant antibodies. Here, "specifically binds" means that the binding affinity to a target substance is superior to other substances to the extent that the presence of the target substance can be detected by binding. In addition, the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of an antibody molecule. The functional fragment of the antibody molecule refers to a fragment that has at least an antigen-binding function, and may be Fab, F(ab'), F(ab')2, Fv, etc. It is preferable that the membrane-bound anti-CD16 antibody according to the present invention is a monoclonal antibody having a single affinity that binds to only one antigenic determinant (epitope). The above membrane-bound anti-CD16 antibody may be a monoclonal antibody clone such as eBioCB16 (CB16), 3G8, B73.1, MEM-154, etc., but is not limited thereto. According to one specific example of the present invention, the monoclonal antibody may be at least one selected from the group consisting of CB16, 3G8, B73.1, and MEM-154. These monoclonal antibodies may have some overlap or differences in the antigenic determinants they bind to. The term "membrane bound interleukin (mbIL)" used in the present invention refers to an interleukin bound to a cell membrane, and may be distinguished from an interleukin secreted extracellularly. mbIL-12 may have a sequence homology of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% to the nucleic acid sequence of SEQ ID NO: 11 or its amino acid sequence. mbIL-18 may have a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% to the nucleic acid sequence of SEQ ID NO: 12 or its amino acid sequence. According to one specific example of the present invention, the culture auxiliary cells may express human leukocyte antigen E (HLA-E) and membrane-bound anti-CD16 antibody (mbCD16). In addition, according to one specific example of the present invention, the culture auxiliary cells may express human leukocyte antigen E (HLA-E), membrane-bound interleukin-12 (mbIL-12), and membrane-bound interleukin-18 (mbIL-18). In addition, according to one specific example of the present invention, the culture auxiliary cells may express human leukocyte antigen E (HLA-E), membrane-bound anti-CD16 antibody (mbCD16), membrane-bound interleukin-12 (mbIL-12), and membrane-bound interleukin-18 (mbIL-18). Composition for activating and amplifying NK cells One aspect of the present invention provides a composition for activating and amplifying NK cells, including the above-described culture auxiliary cells. The specific details of the above culture auxiliary cells are as described above. The composition for activating and amplifying NK cells according to the present invention can induce amplification and / or activation of NK cells by including cultured auxiliary cells expressing at least one selected from the group consisting of membrane-bound anti-CD16 antibody, membrane-bound IL-12, and membrane-bound IL-18 together with HLA-E, and thus can be usefully used for culturing, separating, or proliferating NK cells. The NK cells of the present invention include NK cells without further modification derived from any tissue source, and may include mature NK cells as well as NK progenitor cells. The NK cells are activated in response to interferon or macrophage-derived cytokines and contain two types of surface receptors that control the cytotoxic activity of the cells, labeled as activating receptors and inhibitory receptors. The NK cells may be generated from hematopoietic cells, such as hematopoietic stem or progenitor cells, from any source, such as placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc. According to one specific example of the present invention, the composition may further comprise a cytokine for activating NK cells. The term "cytokine" as used in the present invention refers to a protein (5 to 20 kDa) that plays a role in cell signaling, is released by cells, and affects the behavior of the cells that release the cytokine and / or other cells. The cytokines can be classified functionally, and examples thereof include interleukin (IL), lymphokine, monokine, chemokine, interferon (IFN), hematopoietic factor, growth factor, tumor necrosis factor (TNF) superfamily, adipokine, and neurotrophic factor. These cytokines can be produced by a wide range of cells, including, but not limited to, immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, endothelial cells, fibroblasts, and stromal cells. Furthermore, these cytokines can be produced by more than one type of cell. These cytokines act through receptors and are particularly important in the immune system, regulating the balance between humoral and cellular immune responses, and modulating the maturation, growth, and responsiveness of cell populations. The cytokine of the present disclosure may be a naturally occurring cytokine or a mutant version of a naturally occurring cytokine. "Naturally occurring" herein may also refer to wild-type cytokines, including allelic variants. The term "mutated version of a naturally occurring cytokine" or "mutation" refers to specific mutations made to a naturally occurring sequence that alter the function, activity, and / or specificity of the cytokine. For example, the mutations may enhance or diminish the function, activity, and / or specificity of the cytokine. The mutations may include deletions or additions of one or more amino acid residues of the cytokine. The cytokine may be at least one selected from the group consisting of BMP (Bone morphogenetic protein) family, CCL (Cheomkine ligands) family, CMTM (CKLF-like MARVEL transmembrane domain containing member) family, CXCL (CXC motif ligand ligand) family, GDF (Growth / differentiation factor) family, growth hormone, IFN family, IL family, TNF family, GPI (glycophosphatidylinositol), SLUPR-1 (Secreted Ly-6 / uPAR-Related Protein 1), SLUPR-2 (Secreted Ly-6 / uPAR-Related Protein 2), and combinations thereof. According to one specific example of the present invention, the composition may further comprise interleukin or a mutant thereof as a cytokine. Many interleukins are synthesized by helper CD4 T lymphocytes as well as monocytes, macrophages, and endothelial cells, and can promote the development and differentiation of T and B lymphocytes and hematopoietic cells. More specifically, the interleukin may further include, but is not limited to, one or more selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 and IL-36. Methods for activating and amplifying NK cells One aspect of the present invention provides a method for activating and amplifying NK cells, comprising the step of contacting NK cells with a cultured auxiliary cell or a composition for activating and amplifying NK cells containing the cultured auxiliary cell. The specific details of the above cultured auxiliary cells and the composition for activating and amplifying NK cells including the above cultured auxiliary cells are as described above. The method for activating and expanding NK cells of the present invention is a process of obtaining a sample containing NK cells from an individual, and then mixing and culturing (co-culturing) NK cells or a population thereof with the cultured auxiliary cells or the cultured auxiliary cells included in the composition to stimulate, activate or expand a subpopulation of NK cells. The sample may be a biological sample derived from an individual, for example, a mammal including a human. In addition, the biological sample may be isolated from the individual, and may be, for example, but is not limited to, blood, whole blood, serum, plasma, lymph, urine, feces, tissue, cells, organs, bone marrow, saliva, sputum, cerebrospinal fluid, or a combination thereof. According to one specific example of the present invention, the NK cells may be isolated from a blood sample, for example, whole blood. The whole blood includes peripheral blood mononuclear cells (PBMCs), purified NK cells, and primary resting cells (i.e., isolated directly from blood). As used herein, “stimulation of NK cells” may mean increasing the activity of NK cells, for example, cytotoxic activity, in vitro or in vivo, or causing activated NK cells to be generated, increased, amplified or proliferated. According to one specific example of the present invention, the step may be co-culturing NK cells and culture aid cells. Specifically, the NK cells may be derived from PBMCs, and the cultured feeder cells may be separately treated to inactivate proliferation. According to one specific example of the present invention, the culture auxiliary cells may be irradiated with 50 Gy to 300 Gy. According to one specific example of the present invention, the co-culture is 1 X 10 6 0.1 to 10 X 10 NK cells per 6 It may be to cultivate the culture auxiliary cells. According to one specific example of the present invention, the co-culture may be performed in the presence of cytokines. Specifically, the cytokine may be an interleukin or a mutant thereof, and may further include one or more selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 and IL-36. For example, the cytokines added to the co-culture may be IL-2 and IL-15. Specifically, the co-culture may include culturing in the presence of IL-2 while adding IL-15 to the culture medium. The IL-2 may be used at a concentration of 1 to 500 U / mL, 1 to 400 U / mL, 1 to 300 U / mL, 1 to 200 U / mL, 1 to 100 U / mL, 1 to 50 U / mL, or 5 to 30 U / mL. The IL-15 may be used at a concentration of 1 to 50 ng / mL, 1 to 40 ng / mL, 1 to 30 ng / mL, 1 to 20 ng / mL, or 1 to 10 ng / mL. According to one specific example of the present invention, the co-cultivation may be performed for 2 to 30 days. NK cells that have completed co-culture according to the present invention may be activated. Activated NK cells may refer to cells in which cytotoxicity or the inherent immunoregulatory capacity of NK cells is activated compared to parent cells, for example, hematopoietic cells, or NK progenitor cells. The activated NK cells or a population enriched in activated NK cells can be assessed by detecting at least one functionally relevant marker, for example, CD16, CD57, CD69, CD94, CD161, CD158a, CD158b, NKp30, NKp44, NKp46, DNAM-1,2B4, KIR (e.g., KIR2DL1, KIR2DL2 / 3, KIR3DL1) and the NKG2 family of activating receptors (e.g., NKG2A, NKG2C, NKG2D). The method for activating and amplifying NK cells according to the present invention may further include a step of recovering activated NK cells after the step of contacting the NK cells with a cultured auxiliary cell or a composition for activating and amplifying NK cells containing the cultured auxiliary cell. The recovery of the above NK cells may be performed using any cell recovery method known in the art without limitation. Method for examining the activity of NK cells and method for providing information for diagnosing NK cell-related diseases One aspect of the present invention provides a method for examining the activity of NK cells, comprising the steps of: i) contacting NK cells with the cultured auxiliary cells or the composition to activate NK cells; and ii) analyzing the degree of activation of the activated NK cells. In addition, one aspect of the present invention provides a method for providing information for diagnosing an NK cell-related disease, comprising the steps of: i) contacting NK cells with the cultured auxiliary cells or the composition to activate NK cells; and ii) analyzing the degree of activation of the activated NK cells. The description of the above step i) overlaps with the method for activating and amplifying NK cells described above, and the specific details of the culture auxiliary cells and composition are as described above. In the method for testing the activity of NK cells of the present invention, the NK cells of step i) may exist together with other blood cells or lymphocytes, and may be stimulated after being separated from a blood sample, if necessary. For example, when NK cells exist together with other blood cells or lymphocytes, the separation step may be performed after stimulation, and in order to use only NK cells as lymphocytes, stimulation may be performed after the separation step. The degree of purification of the separated NK cells and the composition of the sample may vary to the extent necessary for the experiment, and are not particularly limited as known in the art. The NK cells may be used as they are purified from the sample, if necessary, or may be used after being expanded to secure conditions or cell amounts suitable for the experiment. However, since a combination of certain factors is specific to NK cells, the step of isolating the NK cells may not be essential when using a combination of factors specific to NK cells in the method of the present invention. The above step ii) is a process of measuring the degree of NK cell activation according to a method known in the art and comparing it with a normal control group. According to one specific example of the present invention, the degree of activation of NK cells in step ii) may be measured by measuring at least one selected from the group consisting of degranulation activity, cytotoxicity activity, and cytokines secreted by NK cell stimulation. The above-mentioned degranulation activity may refer to the induction of target cell lysis, for example, through the secretion of perforin or granzyme, and this can be analyzed using FACS. Specifically, a method can be used to measure CD107a expression, which is proportional to degranulation, after stimulating PBMCs isolated from whole blood samples or purely isolated NK cells, using a fluorochrome-conjugated antibody. The cytotoxic activity can be measured, for example, by culturing a culture containing target cells capable of activating NK cells labeled with a europium fluorescent dye, and then measuring the amount of fluorescent dye released through target cell lysis using a microplate reader. The cytokine secreted by the above NK cell stimulation may be one or more selected from the group consisting of IFN-γ, TNF-α, TNF-β, MIP1-α, MIP-1β, PANTES, IL-8, and IL-10. Analysis of the expression of the immune activator of the NK cell as described above may be performed using FACS, intracellular cytokine staining, ELISA, etc. Specifically, a method may be used to measure the expression of the immune activator in the NK cell by staining the surface of the NK cell using a specific antibody conjugated to fluorochrome, permeabilizing the cell, and staining the cytokine, etc. with another specific immune activator (e.g., IFN-γ) antibody conjugated to fluorochrome. According to one specific example of the present invention, in step ii), the degree of activation of the activated NK cells may be compared with that of normal NK cells. Specifically, when normal NK cells are stimulated with the above-mentioned cultured feeder cells under conditions equivalent to those of the experimental group as a control group, if the activation phenomenon shown in the normal NK cells is significantly higher in the experimental group, or does not appear or the degree is significantly lower, it is judged as abnormal. By this comparison, pathological signs of diseases related to abnormal NK cells, viral infection, the presence of cancer cells, and specific cancers can be judged, and the prognosis for the above diseases can be predicted. The "normal NK cell" refers to an NK cell possessed by or derived from an individual who does not have a disease, and the individual who does not have the disease does not have at least a physical, genetic, or external condition known to affect NK cell activity. In the method for providing information for diagnosing an NK cell-related disease of the present invention, the NK cell-related disease is one that shows abnormal NK cell activity, and may be, for example, a hypersensitive immune disease, an autoimmune disease, an immune rejection reaction, an immunodeficiency disease, a histiocytosis, cancer, type 2 diabetes, a parasitic infection disease, and a viral disease. The hypersensitive immune disease may be at least one selected from asthma and sinusitis, the autoimmune disease may be at least one selected from lupus, multiple sclerosis, type 1 diabetes, and rheumatoid arthritis, or the histiocytosis may be at least one selected from HLH, XLP1, and XLP2. Hemophagocytic lymphohistiocytosis (HLH) may include group II Langerhans cell histiocytosis, erythrophagocytic lymphohistiocytosis (familial, sporadic), infection-associated hemophagocytic syndrome, virus-associated hemophagocytic syndrome, histiocytosis with massive lymphadenopathy, or reticulohistiocytosis. The term "cancer" may include a tumor, a hematological cancer, or a solid cancer, which impairs the synergistic activity of NK cells of the subject or does 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, stomach 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, hematological cancer, lymphoma, skin cancer, psoriasis, and fibroadenoma. In one specific example, the cancer may be pancreatic cancer or B cell lymphoma. In one specific example, the viral disease may be hepatitis B. In one specific example, the immunodeficiency disease may be DiGeorge syndrome or Chediak-Higashi syndrome. Information on the above NK cell activity-related disease can be determined as an abnormal NK cell if no activation of the NK cells of the experimental group is detected compared to normal NK cells, or can be determined as being related to a specific disease if NK cells that have lost activity for a specific receptor abnormally or do not cause activity for a target cell. NK cells, cell therapy agents, pharmaceutical compositions, uses of NK cells, and methods for treating diseases One aspect of the present invention provides NK cells manufactured by a method for activating and amplifying the NK cells. In addition, one aspect of the present invention provides a cell therapy agent comprising the NK cell as an active ingredient. In addition, one aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer or infectious disease, which comprises the NK cell as an active ingredient. In addition, one aspect of the present invention provides a use of the NK cells for the manufacture of a medicine. In addition, one aspect of the present invention provides a disease treatment method for preventing or treating cancer or infectious disease, comprising a step of administering the NK cells to an individual. According to one specific example of the present invention, the NK cells may include NK cells or a population thereof. As used herein, “disease” may mean a pathological condition, particularly cancer, infectious disease, inflammatory disease, metabolic disease, autoimmune disorder, degenerative disease, apoptosis-related disease, and graft rejection. As used herein, “treatment” refers to or includes alleviating, inhibiting the progression of, or preventing a disease, disorder, or condition, or one or more symptoms thereof, and “active ingredient” or “pharmaceutically effective amount” may mean any amount of a composition used in the course of practicing the invention provided herein that is sufficient to alleviate, inhibit the progression of, or prevent a disease, disorder, or condition, or one or more symptoms thereof. As used herein, "administering," "introducing," and "transplanting" are used interchangeably and may refer to the placement of a composition according to one embodiment into a subject by a method or route that results in at least partial localization of the composition to a desired site. Administration may be by any suitable route that delivers at least a portion of the cells or cellular components of the composition according to one embodiment to a desired location within a viable subject. The survival period of the cells after administration to a subject may be as short as several hours, for example, 24 hours, to several days, or as long as several years. As used herein, “isolated cell”, such as “isolated immune cell”, means a cell that is substantially separated from the tissue from which the cell originates, such as a hematopoietic cell. In one embodiment, the method of administration of the pharmaceutical composition is not particularly limited, but may be administered parenterally, such as intravenously, subcutaneously, intraperitoneally, by inhalation, or topically, or may be administered orally, depending on the intended method. The dosage may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage refers to the amount of a therapeutic substance according to one aspect that is sufficient to treat the alleviated disease state by being administered to a subject in need of treatment. The effective amount of a therapeutic substance varies depending on the specific compound, the disease state and its severity, and the subject in need of treatment, and this can be routinely determined by a person skilled in the art. As a non-limiting example, the dosage of the composition according to one aspect to a human may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity. For example, based on an adult patient weighing 70 kg, the dose may be approximately 1,000 to 10,000 cells / time, 1,000 to 100,000 cells / time, 1,000 to 1,000,000 cells / time, 1,000 to 10,000,000, 1,000 to 100,000,000 cells / time, 1,000 to 1,000,000,000 cells / time, 1,000 to 10,000,000,000 cells / time, or divided into one or several doses per day at regular intervals, or administered multiple times at regular intervals. The term "subject" as used in the present invention means a subject requiring treatment of a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow. According to one specific embodiment of the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier and / or additive. For example, it may include sterile water, physiological saline, a conventional buffer (such as phosphoric acid, citric acid, or other organic acids), a stabilizer, a salt, an antioxidant (such as ascorbic acid), a surfactant, a suspending agent, an isotonic agent, or a preservative. For topical administration, it may also include a combination with an organic substance such as a biopolymer, 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 pharmaceutical composition is prepared in a dosage form suitable for injection, the immune cells, immune cells, or a substance that increases their activity may be dissolved in a pharmaceutically acceptable carrier or may be frozen in a dissolved solution state. According to one specific example, the pharmaceutical composition may appropriately contain, if necessary, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc. depending on the administration method or formulation. 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 specific example may be manufactured in a unit dose form or may be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets, or capsules. The NK cell culture auxiliary cell according to the present invention stimulates NK cells by expressing one or more of mbCD16, mbIL-12 and mbIL-18 together with HLA-E, thereby increasing the activation and expansion of NK cells, and such NK cells can be used as a cell therapeutic agent and a pharmaceutical composition for preventing or treating cancer or infectious diseases. Figure 1 shows the HLA-E expression levels of K562 (K562-HLA-E) expressing HLA-E, K562 (K562-HLA-E UL40F) expressing HLA-E and UL40F, and K562 (K562-HLA-E UL40I) expressing HLA-E and UL40I as culture auxiliary cells according to one embodiment of the present invention. Figure 2 shows the expression levels of mbCD16-1 or mbCD16-2 of K562 (K562-HLA-E-mbCD16-1) expressing HLA-E and mbCD16-1, and K562 (K562-HLA-E-mbCD16-2) expressing HLA-E and mbCD16-2 as culture auxiliary cells according to one embodiment of the present invention. FIG. 3 shows the expression levels of mbCD16-1 or mbCD16-2 of K562 (K562-HLA-E UL40F-mbCD16-1) expressing HLA-E and mbCD16-1, and K562 (K562-HLA-E UL40F-mbCD16-2) expressing HLA-E and mbCD16-2 as culture auxiliary cells according to one embodiment of the present invention. FIG. 4 shows the expression levels of mbCD16-1 or mbCD16-2 of K562 (K562-HLA-E UL40I-mbCD16-1) expressing HLA-E and mbCD16-1, and K562 (K562-HLA-E UL40I-mbCD16-2) expressing HLA-E and mbCD16-2 as culture auxiliary cells according to one embodiment of the present invention. FIG. 5 shows the expression levels of mbIL-12 and mbIL-18 of K562 (K562-HLA-E UL40F-mbCD16-1-mbIL12 / 18) expressing HLA-E, mbCD16-1, mbIL-12, and mbIL-18 as a culture auxiliary cell according to one embodiment of the present invention. Figure 6 shows the level of gNK expression co-cultured with cultured auxiliary cells according to one embodiment of the present invention, with A and B using PBMCs from different donors. Figure 7 shows the cell killing ability of gNK co-cultured with culture auxiliary cells according to one embodiment of the present invention. Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples. Example 1. Production of cultured auxiliary cells K562 (human myelogenous leukemia cell line) was obtained from the American Type Culture Collection (ATCC). The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin (Invitrogen) at 37°C in a humidified 5% CO2 incubator. K562 cells were then used to produce feeder cells. 1-1. Production of HLA-E-expressing cultured feeder cells K562 expressing HLA-E was produced, and the CMV peptide UL40F or UL40I was additionally expressed to promote the expansion of NK cells. First, the human HLA-E gene (SEQ ID NO: 1), UL40F (SEQ ID NO: 2), or UL40I (SEQ ID NO: 3) was cloned into the lentiviral vector pCDH-CMV-EF1-GFP to construct recombinant lentivirus production vectors Vector_HLA-E (SEQ ID NO: 4), Vector_HLA-E-UL40F (SEQ ID NO: 5), and Vector_HLA-E-UL40I (SEQ ID NO: 6). Then, for virus production, each of the constructed vectors was transfected into 293FT cells together with the packaging vector using lipofectamin3000 (Invitrogen). After a period of time, the medium was replaced with fresh medium, and the cells were cultured for 48 hours, and then the medium containing the virus was collected. The recovered medium was centrifuged at 500 x g for 10 minutes, and only the pure medium containing the virus was separated using a 0.45 ㎛ filter to produce HLA-E, HLA-E + UL40F, or HLA-E + UL40I expressing lentiviruses. Then, 1 mL of each lentivirus was dissolved in 9 mL of medium containing K562 and added together with polybrene (8 ㎍ / ml), and cell culture was performed for 48 hours. Thereafter, infected cells were selected using flow cytometry (fluorescence-activated cell sorting, FACS). Cell surface proteins and intracellular proteins were stained for flow cytometry analysis. The purity of NK cells and gNK cells was confirmed by the expression of CD3-CD56+ and CD56+FcRγ- / CD3-CD56+, respectively. First, in cell surface protein staining, about 1 X 10 6The cells were transferred to a 96-well U-bottom plate, centrifuged, and the supernatant was removed, leaving only the cells. 1 ul each of the antibodies for the surface proteins to be analyzed was added to 100 ul of FACS buffer (1% FBS in PBS): CD3 (APC-Cy7) (Invitrogen, 47-0036-42), CD56 (PE-Cy7) (Invitrogen, 25-0567-42), CD57 (APC) (Biolegend, 359610), and NKG2C (BV421) (BD, 748169). 100 ul of FACS buffer containing each antibody was added to the 96-well plate containing the cells, and staining was performed for 15 minutes in the dark at 4°C. After washing with FACS buffer, the supernatant was removed, leaving only the cells, and centrifuged. For intracellular protein staining, the Fixation / Permeabilization Kit (BD, 554714) was used. Cells were treated with 100 μl of 1X Perm / Wash buffer containing 1 μl of FcεRI (γ subunit-FITC) (Merck, FCABS400F), and stained for 30 min in the dark at 4℃. After washing with 1X Perm / Wash buffer, the supernatant was removed by centrifugation. After adding 200 μl of 1X Perm / Wash buffer to the cells, flow cytometry analysis was performed. The cultured auxiliary cells expressed reporter genes such as GFP and RFP, so no separate staining was performed. As a result, referring to Fig. 1, among the selected cells, K562 expressing HLA-E was named 'K562-HLA-E', ​​and K562 expressing UL40F or UL440I together with HLA-E was named 'K562-HLA-E UL40F' and 'K562-HLA-E UL40I', respectively. 1-2. Production of HLA-E and mbCD16-expressing cultured feeder cells CD16 is a key factor that promotes the selective expansion of gNK cells lacking FcεRIγ among NK cells. We produced cultured feeder cells expressing membrane-bound anti-CD16 antibodies (mbCD16-1 or mbCD16-2) together with HLA-E. First, the monoclonal antibody 3G8 clone of anti-CD16 antibody, mbCD16-1 (SEQ ID NO: 7) or mbCD16-2 (SEQ ID NO: 8), was cloned into the lentiviral vector pCDH-CMV-EF1-RFP to construct vectors for producing recombinant lentiviruses, Vector_mbCD16-1 (SEQ ID NO: 9) and Vector_mbCD16-2 (SEQ ID NO: 10). Then, for virus production, each of the constructed vectors was transfected into 293FT cells together with the packaging vector using lipofectamin3000 (Invitrogen). After a period of time, the medium was replaced with fresh medium, and the cells were cultured for 48 hours, after which the medium containing the virus was collected. The recovered medium was centrifuged at 500 xg for 10 minutes, and only the pure medium containing the virus was separated using a 0.45 ㎛ filter to produce mbCD16-1 or mbCD16-2 expressing lentivirus. Thereafter, 1 mL of each lentivirus was dissolved in 9 mL of the medium containing K562-HLA-E, K562-HLA-E UL40F, or K562-HLA-E UL40I produced in Example 1-1, and added together with polybrene (8 ㎍ / ml), and cell culture was performed for 48 hours. Thereafter, infected cells were selected using flow cytometry. Referring to Figures 2 to 4, among the selected cells, K562 expressing HLA-E and mbCD16-1 were named 'K562-HLA-E-mbCD16-1', 'K562-HLA-E UL40F-mbCD16-1', and 'K562-HLA-E UL40I-mbCD16-1', and K562 expressing HLA-E and mbCD16-2 were named 'K562-HLA-E-mbCD16-2', 'K562-HLA-E UL40F-mbCD16-2', and 'K562-HLA-E UL40I-mbCD16-2'. 1-3. Production of HLA-E, mbCD16, mbIL-12, and mbIL-18-expressing cultured feeder cells gNK cells have reduced expression of receptors for cytokines IL-12 or IL-18, which may weaken their responsiveness to IL-12 or IL-18 during NK cell amplification. Therefore, to provide cytokine stimulation to supplement memory function during the amplification phase, we created cultured feeder cells expressing membrane-bound forms of IL-12 and IL-18 (mbIL-12 / 18) together with HLA-E and mbCD16. First, mbIL-12 (SEQ ID NO: 11) and mbIL-18 (SEQ ID NO: 12) were cloned into the lentiviral vector pCDH-CMV-EF1-GFP to construct a vector for producing recombinant lentiviruses, Vector_mbIL12 / 18 (SEQ ID NO: 13). Then, for virus production, the constructed vectors were transfected into 293FT cells together with the packaging vector using lipofectamin3000 (Invitrogen). After a period of time, the cells were cultured for 48 hours after replacing the medium with fresh medium, and the medium containing the viruses was collected. The collected medium was centrifuged at 500 × g for 10 minutes, and only the pure medium containing the viruses was separated using a 0.45 ㎛ filter to produce mbIL-12 and mbIL-18 expressing lentiviruses. Afterwards, 1 mL of the lentivirus was dissolved in 9 mL of medium containing K562-HLA-E UL40F-mbCD16-1 produced in Example 1-2, and added together with polybrene (8 μg / ml), and cell culture was performed for 48 hours. Afterwards, infected cells were selected using flow cytometry, and the expression of mbIL-12 and mbIL-18 in the selected cells was confirmed using RT-qPCR (Quantitative reverse transcription PCR) (Fig. 5). Among the selected cells, K562 expressing HLA-E, mbCD16-1, mbIL-12, and mbIL-18 was named 'K562-HLA-E UL40F-mbCD16-1-mbIL12 / 18'. For PCR, cultured feeder cells were lysed and mRNA was extracted. Using 1 mg of extracted mRNA, 1 mg of cDNA was synthesized, which served as a template for amplifying mbIL-12 and mbIL-18. PCR was performed at 60°C for 30 cycles using the primers listed in Table 1 below. Primer name Primer sequence (5'-3') Sequence number mbIL18_FGAA TGA CCA AGT TCT CTT CAT TGA CCA AG14 mbIL18_RGTT CTC ACA GGA GAG AGT TGA AAT TTT CTC15 mbIL12_FGAT GCC GTT CAC AAG CTC AAG16 mbIL12_RGAA CGC AGA ATG TCA GGG AGA AGT AG17 Example 2. Phenotypic characteristics of NK cells amplified by cultured feeder cells. Ficoll-Hypaque (d = 1.077, Lymphoprep TM) from healthy adult donors

[0076] ; Human peripheral blood mononuclear cells (PBMC) were separated using Axis-Shield, Oslo, Norway, and washed twice with PBS. The cultured feeder cells of Example 1 were prepared by irradiating each with 100 Gy of gamma rays. For co-culture of cultured feeder cells and PBMC, the cultured feeder cells (2.55 x 10) of Example 1 were placed in a 24-well plate. 5 cells / well) and PBMC (5 x 10 5(cells / well) were cultured in RPMI 1640 medium (containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, and 4 mmol / L L-glutamine) supplemented with 10 U / mL IL-2, 10 U / mL IL-12, and 100 ng / mL IL-18. IL-2 and IL-18 were not added in co-cultures with feeder cells expressing membrane-bound IL-12 and IL-18. Gamma-irradiated feeder cells were added for re-stimulation on days 7 and 14. The medium was changed every 2–3 days using RPMI 1640 medium containing 200 U / mL IL-2 and 5 ng / mL IL-15, and cultured for up to 21 days. Phenotypic characteristics of the expanded NK cells were confirmed using flow cytometry on days 7, 14, and 21 of culture. As a result, as shown in Fig. 6, when HLA-E was expressed in the cultured feeder cells, the expression level of gNK cells among the expanded NK cells was higher than when HLA-E was not expressed, and the expression level of gNK cells increased as mbCD16-1 / 2 was additionally expressed. In particular, the expression level of gNK cells was the highest when co-cultured with cultured feeder cells expressing all of HLA-E, mbCD16-1, and mbIL-12 / 18. Example 3. ADCC Evaluation of NK Cells Amplified by Cultured Feeder Cells gNK lacks FcεRIγ and thus has lower cytotoxicity than conventional NK cells. However, when gNK binds to anti-CD16 antibodies, the CD16 signal is strengthened, resulting in enhanced ADCC function. Therefore, to evaluate the ADCC of gNK amplified by the K562-HLA-E UL40F-mbCD16-1-mbIL12 / 18 cultured feeder cells of Example 1, the cell killing ability was analyzed. First, Raji cells were stained with 0.5 μM CFSE in FACS buffer at 37°C for 10 minutes and then washed twice with RPMI medium. The CFSE-stained Raji cells were coated with 10 μg / mL rituximab for 3 minutes at room temperature. On day 14 of culture, gNK cells and Raji cells were mixed in 96-well U-bottom plates at an E:T ratio (2:1, 1:1, 0.5:1). The plates were centrifuged at 400 × g for 3 minutes and incubated in a 5% CO2 incubator for 4 hours. After incubation, the cells were stained with 1 mg / mL propidium iodide and analyzed for apoptosis using flow cytometry. As a result, as shown in Fig. 7, gNK obtained on the 14th day of co-culture with the cultured feeder cells K562-HLA-E UL40F-mbCD16-1-mbIL12 / 18 exhibited cell killing ability against the lymphoma cell line Raji, and when the antibody rituximab targeting CD20 was added during co-culture, the ADCC of gNK further increased. The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. human leukocyte antigen E; and A feeder cell for activating and expanding NK cells expressing at least one selected from the group consisting of membrane bound anti-CD16 antibody, membrane bound interleukin-12, and membrane bound interleukin-18.

2. In claim 1, The above culture auxiliary cells are culture auxiliary cells selected from the group consisting of K562, RPMI8866, ARH77, EBV_LCL, 721.221, and HFWT cells.

3. In claim 1, The above membrane-bound anti-CD16 antibody is a culture feeder cell selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant antibodies and fragments thereof.

4. In claim 3, The above monoclonal antibody is a culture assistant cell selected from the group consisting of CB16, 3G8, B73.1 and MEM-154.

5. In claim 1, The above-mentioned cultured feeder cells are cultured feeder cells expressing human leukocyte antigen E, membrane-bound anti-CD16 antibody, membrane-bound interleukin-12 and membrane-bound interleukin-18.

6. A composition for activating and amplifying NK cells, comprising a culture auxiliary cell of any one of claims 1 to 5.

7. In claim 6, A composition further comprising a cytokine.

8. A method for activating and amplifying NK cells, comprising the step of contacting NK cells with the culture auxiliary cell of claim 1 or the composition of claim 6.

9. In claim 8, A method wherein the above NK cells are isolated from a blood sample.

10. In claim 8, The above step is a method of co-culturing NK cells and culture feeder cells.

11. In claim 10, A method wherein the above cultured auxiliary cells are irradiated with 50 Gy to 300 Gy.

12. In claim 10, The above coculture is 1 X 10 6 0.1 to 10 X 10 NK cells per 6 A method for culturing auxiliary cells.

13. In claim 10, A method wherein the above co-culture is performed in the presence of cytokines.

14. In claim 13, The cytokines include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36. 15.i) a step of activating NK cells by contacting the culture auxiliary cell of claim 1 or the composition of claim 6 to NK cells; and ii) A method for examining the activity of NK cells, comprising a step of analyzing the degree of activation of the activated NK cells.

16. In claim 15, A method wherein the degree of activation of NK cells in step ii) is measured by measuring at least one selected from the group consisting of degranulation activity, cytotoxic activity, and cytokines secreted by NK cell stimulation. 17.i) a step of activating NK cells by contacting the culture auxiliary cell of claim 1 or the composition of claim 6 to NK cells; and ii) A method for providing information for diagnosing a NK cell-related disease, comprising a step of analyzing the degree of activation of the activated NK cells.

18. In claim 17, A method wherein the degree of activation of NK cells in step ii) is measured by measuring at least one selected from the group consisting of degranulation activity, cytotoxic activity, and cytokines secreted by NK cell stimulation.

19. NK cells manufactured by the method for activating and amplifying NK cells of claim 8.

20. A cell therapy agent comprising NK cells of claim 19 as an effective ingredient.

21. A pharmaceutical composition for preventing or treating cancer or infectious disease, comprising the NK cell of claim 19 as an effective ingredient.

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