KR20180033095A - A novel Natural Killer cell line and use thereof - Google Patents

Abstract

The present invention relates to a novel natural killer cell line and a use thereof, and more specifically, to a novel natural killer cell line in which CD2, CD11a, CD25, CD45, CD54, CD56 and HLA-DR are positive; and CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, TCRαβ and TCRγδ are negative. The present invention also relates to a pharmaceutical composition for treating cancer including the natural killer cell line, and uses thereof.

Description

A novel natural killer cell line and use thereof

The present invention relates to a novel cell line and its use, specifically to a novel natural killer (NK) cell line and its use.

Currently, the basic direction of cancer immunotherapy that is currently being carried out is the adoption of immune cells derived from autologous cancer patients to proliferate and activate cancer-specific immune cells in vitro, cell therapy (ACT) technology. The main immune cells used for the treatment of cancer cells are mainly cytotoxic T lymphocytes (CTL), dendritic cells (DC), natural killer cells 'NK'), the most widely used method since cell therapy with T-cells derived from patients was first introduced by Dr. Steven Rosenberg of the NIH in 1985.

T cell therapy has been developed up to the third generation until now. First generation T cell therapy is a method of proliferating all T cells (bulk T cells) present in blood or cancer tissues and administering them to patients. The effect could not be expected. In the case of the second-generation T-cell therapy, which is complementary to the above-described second-generation T-cell therapeutic agent, Ag-specific T cells were isolated and mass-cultured for administration to cancer patients. There is a problem that the process is long and complicated. The third generation T cell therapy consists of 1) introducing a TCR gene that recognizes a specific cancer antigen directly into a T cell or 2) introducing a T cell activation domain (T cell) into an antigen recognition site (scFv) of a monoclonal antibody recognizing a specific cancer antigen In addition to enhancing antigen specificity and shortening the production period by introducing a chimeric antigen receptor (hereinafter, referred to as 'CAR') coupled with an activation domain into T cells, the therapeutic effect is also excellent, and some leukemia and lymphoma (Rosenberg et al ., Nat. Rev. Cancer 8: 299-308, 2008).

Despite these encouraging results, CART therapy with T cells still has some limitations (Challice L Bonifant, 2016, Molecular Therapy). First, in performing CART (chimeric antigen receptor T cell) or ACT, it is difficult to perform the treatment immediately because of the long cycle of isolating and purifying the blood cells of the patient and amplifying the blood cells. In addition, The cost of treatment is significantly higher than with conventional therapy. Second, the immune cells present in the patient after chemo-chemotherapy are significantly less in function than the immune cells in the healthy patient, and the number of immune cells available due to the rapid decrease in the number of blood cells after chemotherapy Also less. Third, the CART cell-mediated method can induce cytokine release syndrome such as IL-6, bystander activation of existing TCR, neurological toxicity, allograft graft-versus-host disease (GVHD), leading to serious side effects. Therefore, to overcome these disadvantages, there is an alternative to allogenic immune cells for cell co-cultivation, the most likely being NK cells.

NK cells have the ability to kill abnormal autologous cells, or cancer cells, similar to cytotoxic T cells (CTLs). NK cells are characterized by the absence of specificity for antigen, unlike T cells expressing antigen-specific antigen receptors, and the presence of NK activation receptor (KAR) and killer cell inhibitory receptor (KIR) (Cheng et al ., Cell Mol. Immunol ., 10: 230-252, 2013), which recognize abnormal changes in cells such as expression and absence of surface MHC I antigen. In particular, NK cells are considered to be suitable cells for ACT because they eliminate leukemia cells and cancer cells without causing graft-versus-host disease (Glienke et al ., Front. Pharmacol ., 6: Article 21, 2015). In addition, NK cells do not have immune memory, unlike T cells, and are killed by cancer cells after they are activated. Therefore, the chemotherapy with NK cells is very unlikely to have side effects in comparison with the long-term T cell-based therapies that form immune memory in the body. However, in spite of the possibility of NK cell itself, the method of producing a therapeutic agent by amplifying a patient's self-derived cell or allogeneic NK cell by simple separation is limited due to limitation of production amount due to limited cell resources, complexity of production process, There may be batch to batch variation resulting from the heterogeneity of the therapeutic agent itself, since it is not a delayed onset, therapeutic agent for single cell origin. In order to overcome these limitations, the development of a cell therapy agent based on a single NK cell line may be a more effective method, thereby simplifying the production process of the cell therapy agent, reducing the difference between the therapeutic agent production time and the administration time point, A cell therapeutic agent can be prepared.

Currently, there are 7 types of NK cell lines constructed globally, of which NK-92 is the only cell line used for clinical trials (see Table 1). In particular, attempts to increase the cancer-specific killing effect by introducing CAR into the NK-92 cell line have existed in the experimental and non-clinical stages, and the clinical safety has been confirmed (Cheng et al ., Cell Mol. Immunol . , 10: 230-252, 2013). However, it has been reported that NK-92 alone does not exhibit sufficient anticancer efficacy, and that it is eliminated in 48 hours after in vivo administration (Tonn et al ., Cytotherapy , 15 (12): 1563-1570, 2013).

Previously established NK cell lines and characteristics name Establishment year Nationality disease origin Phenotype EBV infection Other YT 1985 Japan Acute lymphocytic leukemia and thymoma Pericardial fluid unidentified + The first established NK cell line NK92 1994 Canada Non-Hodgkin
Lymphoma Pericardial fluid CD56 ⁺ CD2 ⁺ CD7 ⁺ CD3 ^- - The only cell line that entered clinical trials NKL 1996 United States of America Giant granulocytic leukemia Pericardial fluid CD56 ^{< / RTI > dim} CD16 ^- CD2 ⁺ CD3 ^- - NK92 and other anti-cancer spectrum HANK-1 1998 Japan NK / T lymphoma Lymph node
→
SCID mouse CD56 ⁺ CD2 ⁺ CD3e ⁺ HLA-DR ⁺ + Type 2 EBV latency
(LMP-1 ⁺ EBNA2 ^-) NK-YS 1998 Japan NK cells
Lymphoma Skin lesion CD56 ⁺ CD2 ⁺ CD5 ⁺ CD3 ^- CD16 ^- + The mouse stromal cell line (SPY32) and co-culture KHGY-1 2000 Japan Aggressive NK
leukemia Peripheral blood CD56 ⁺ CD8a ⁺ CD33 ⁺ CD3 ^- CD16 ^- CD25 ^- - - p53 mutation
- greater cytotoxicity than NK92 cells NKG
(Clinical preparation) 2011 China Non-Hodgkin
Lymphoma Peripheral blood CD56 ^{& lt ; / RTI & gt ; bright} CD2 ⁺ CCR7 ⁺ CXCR4 ⁺ CD3 ^- CD16 ^- - - Prolonged growth period than NK92
- greater cytotoxicity than NK92 cells

It is an object of the present invention to provide new NK cells originating from patient-derived lymphoma.

Another object of the present invention is to provide a recombinant NK cell into which the polynucleotide encoding one or more foreign proteins is introduced into the isolated NK cell.

It is still another object of the present invention to provide a pharmaceutical composition for treating cancer comprising the NK cell line or recombinant NK cell as an active ingredient.

According to one aspect of the present invention there is provided an isolated human NK cell having the following characteristics:

CD2, CD11a, CD25, CD45, CD54, CD56 and HLA-DR are positive;

CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.

According to another aspect of the present invention, there is provided a method of producing a recombinant NK cell, comprising the step of transfecting the isolated NK cell with a polynucleotide encoding one or more exogenous proteins, wherein the polynucleotide is operably linked to a promoter, Recombinant NK cells are provided.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating and preventing cancer comprising the isolated NK cell and / or the recombinant NK cell as an active ingredient.

According to another aspect of the present invention there is provided a method of treating said cancer-bearing organism comprising administering a therapeutically effective amount of said isolated NK cells and / or recombinant NK cells to a subject having cancer .

FIG. 1A is a graph showing the degree of cell proliferation according to the passage of the NK101 cell line of the present invention. FIG. 1B is a photograph of a cell type observed by culturing the NK101 cell using a microscope. FIG. 1D is a dot graph showing the results of flow cytometry on CD3 and CD16, which are the main cell markers of the NK101 cells, FIG. 1E is a graph showing the IL- 2 dependent graphitizing ability.
FIG. 2A is a graph (right side) showing the cell division potential according to the culture medium of the NK101 cell line of the present invention as a cumulative population doubling level (left) and cell survival rate (right) (Left) and a cell survival rate (right) in the same medium of the NK-92 cell line, which is a conventionally established NK cell line.
3 is a histogram showing the results of flow cytometry analysis on T / NK markers in NK101 cells of the present invention.
Fig. 4 is a histogram showing the results of flow cytometry analysis on the major system and ancestral markers in NK101 cells of the present invention.
FIG. 5A is a histogram showing the results of performing flow cytometry on NK cell activation receptors in NK101 cells of the present invention, and FIG. 5B is a histogram showing results of flow cytometry analysis of inactivating NK cell receptors in NK101 cells of the present invention FIG. 5C is a histogram showing the results of flow cytometry analysis of the TK cell adhesion and NK cell activation assisting receptors in NK101 cells of the present invention. FIG.
FIG. 6 is a histogram showing the results of flow cytometry analysis on the expression of perforin, IFN-y and TRAIL, which are involved in cytotoxicity and immunological activation of NK cells in NK101 cells of the present invention.
7 is a histogram showing the results of flow cytometry analysis on cytokine receptors in NK101 cells of the present invention.
8A is a histogram showing the results of flow cytometry analysis on the expression of various CC chemokines (CCR) in the NK101 cell line of the present invention, and FIG. 8B is a histogram showing the results of various CXC chemokine (CXCR) It is a histogram showing the results of flow cytometry analysis.
FIG. 9A is a graph showing anticancer effects against various human cancer cell lines according to the cell mixing ratio of the NK101 cell line of the present invention. FIG. 9B is a graph comparing anticancer effects of NK101 and NK-92 in THP-1 cancer cells, FIG. 9c is a graph comparing the concentrations of INF-γ, an effector molecule observed in culture when co-cultured in NK101 and NK-92 cells, FIG. 9d is a graph comparing the concentrations of INF- FIG. 2 is a graph showing the results of comparing the anti-cancer effect of NK101 with that of NK-92 cells in the model.
FIG. 10A is a schematic diagram showing the structure of a TRAIL expression construct prepared to transform the NK101 cell line of the present invention, and FIG. 10B shows an anti-cancer effect of the recombinant NK101 cell transfected with the TRAIL expression construct It is a histogram showing the result of flow cytometry analysis.
FIG. 11A is a schematic diagram showing the structure of the CD7-CD28 expression construct produced to transform the NK101 cell line of the present invention, and FIG. 11A is a schematic diagram showing the structure of the recombinant NK101 cell transfected with the CD7- FIG. 11C is a graph comparing the cancer cell imaging ability of various cancer cells of the recombinant NK101 cells transfected with the control NK101 cells and the CD7-CD28 expression construct .
FIG. 12A is a graph showing the cell survival rate with time of irradiation of the NK101 cell line of the present invention, and FIG. 12B is a graph showing the cytotoxicity of co-culturing with HCT116 cells according to the dose of radiation to be.

According to one aspect of the present invention there is provided an isolated human NK cell having the following characteristics:

CD2, CD11a, CD25, CD45, CD54, CD56 and HLA-DR are positive;

CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, TCRαβ and TCRγδ are negative.

The isolated NK cell may further comprise the following features in addition to the phenotype:

CD18, CD33, CD122, CD132, CD159 and FAS are positive;

High expression of INF gamma upon IL-2 treatment; And

CD7, CD10, CD11c, CD13, CD19, CD127, CD158a (KIR2DL1), CD158b (KIR2DL2), NKG2C, and ILT2 are negative.

The isolated NK cell may additionally have the following characteristics:

CD94, DNAM1, 2B4, NKp30, NKp46 and NKG2D;

NKp44, NKp80, KIR2DL1 and KIR2DL2 / DL3;

CCR4, CCR6, CCR7, CCR8, CXCR3, and CXCR4 are expressed; And

CCR1, CCR2, CCR3, CCR5, CCR9, CXCR1, CXCR2, CXCR5, CXCR6 and CXCR7 are not expressed.

According to another aspect of the present invention, there is provided a method of producing a recombinant NK cell, comprising the step of transfecting the isolated NK cell with a polynucleotide encoding one or more exogenous proteins, wherein the polynucleotide is operably linked to a promoter, Recombinant NK cells are provided.

In the recombinant NK cell, the exogenous protein may be any protein as long as it is for enhancing the immunity of NK cells. Such an exogenous protein may include a protein specifically binding to a cancer cell-specific receptor or ligand for cancer cell targeting, An immunomodulatory polypeptide comprising a costimulation domain, a chemokine receptor, or an apoptosis-inducing ligand.

As used herein, the term "cancer cell specific receptor or ligand" refers to a cell surface receptor or ligand that is specifically expressed in cancer cells. Such cancer cell specific receptor or ligand includes epithelial growth factor receptor (EGFR), somatostatin receptor (SSTR), α _v β ₅ integrin, vascular endothelial growth factor receptor (VEFGR), human epithelial growth factor receptor 2 (HER2), androgen receptor (AR), estrogen receptor (ER), progesterone receptor (PR) (PD-1L), MUC1, MUC2, MUC3, folate receptors, ErbB2, transferrin receptors, TAG-1 receptors, bombesin receptors, prostate-specific G- 72, G _M3 , Le ^x , CD10, CD20, or CEA.

The protein specifically binding to the cancer cell-specific receptor or ligand may include an antibody specifically binding to the cancer cell-specific receptor or ligand, a functional fragment thereof or an antibody analogue, and the cancer cell-specific receptor or ligand Specific binding protein, for example, a protein having an RGD domain that specifically binds to an integrin that is specifically expressed in cancer cells.

As used herein, the term " antibody "refers to a Y-shaped protein produced from plasma cells used by the immune system to identify or neutralize exogenous substances, such as bacteria or viruses, also referred to as immunoglobulins. The antibody used in this document includes various "functional fragments ", such as Fab, F (ab ') 2, Fab', ScFv and sdAb, from an antibody.

The term "functional fragment of an antibody " used in the present specification includes all of the fragments generated by recombinant methods, as well as fragments obtained by digesting an antibody with a protein cleaving enzyme.

As used herein, the term "Fab" is an antigen-binding antibody fragment that is produced by digesting an antibody molecule into a protease, papain, and is a dimer of two peptides of VH-CH1 and VL- , And another fragment generated by papain is referred to as Fc (fragment crystallizable).

As used herein, the term "F (ab ') 2" refers to a fragment produced by digesting an antibody with protease, pepsin,

Quot; refers to a fragment containing a binding site and a form of a tetramer in which two Fabs are linked by a disulfide bond. Pepsin

The other fragment generated by this is called pFc '.

The term "Fab ", as used herein, is a molecule similar in structure to Fab produced by the separation of F (ab ') 2 under weakly reducing conditions.

As used herein, the term "ScFv" is an abbreviation of "single chain variable fragment ", which is not a fragment of an actual antibody. The heavy chain variable region (VH) and the light chain variable region (VL) (Glockshuber et al., Biochem. 29 (6): 1362-1367, 1990), although it is not a unique antibody fragment as a kind of fusion protein prepared by linking with a linker peptide of the size

The term " sdAb (single domain antibody) ", as used herein, refers to an antibody fragment consisting of a single variable region fragment of an antibody, referred to as a nanobody. The sdAb derived mainly from the heavy chain is used, but a single variable region fragment derived from the light chain has also been reported to be a specific binding to the antigen.

As used herein, the term "antibody mimetic" is intended to mean that, unlike conventional full-length antibodies, in which two heavy chains and two light chains form a quaternary structure of a heterozygous complex, Chain variable fragment (scFv), which is an artificial fragment linked by a linker to a variable region of Fab, F (ab ') 2, Fab' or heavy and light chains, Like protein produced from non-antibody-derived protein scaffolds such as antibody fragments (VHH, VNAR, etc.) derived from camel or cartilaginous fish consisting only of heavy chain or nanobody, monobody, variable lymphocyte receptor (VLR).

As used herein, the term "costimulatory domain" refers to a cytoplasmic domain responsible for the T cell-assisted stimulatory function of the costimulatory factor, an immunological protein that assists T / NK activation it means. These auxiliary stimulatory domains include CD28, inducible costimulator (ICOS), cytotoxic T lymphocyte associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), BTLA (B and T lymphocyte associated protein), DR3 1BB, CD2, CD40, CD30, CD27, SLAM (signaling lymphocyte activation molecule), 2B4 (CD244), NKG2D / DNAX- activating protein 12, TIM1 immunoglobulin and mucin domain containing protein 1), TIM2, TIM3, TIGIT, CD226, CD160, lymphocyte activation gene 3, B7-1, B7-H1, glucocorticoid-induced TNFR family related protein, HVEM mediator) or the cytoplasmic domain of OX40L [ligand for CD134 (OX40), CD252], or a linker of two or more of these.

In the recombinant NK cells, the immunomodulatory polypeptide is selected from the group consisting of CD28, ICOS, CTLA4, PD1, BTLA, DR3, 4-1BB, CD2, CD40, CD30, CD27, SLAM, 2B4, NKG2D) / DAP12, TIM1, TIM2 But are not limited to, TIM3, TIGIT, CD226, CD160, LAG3, B7-1, B7-H1, GITR, HVEM or OX40L or fragments thereof comprising the accessory stimulatory domain (Chen, L. and Flies , DB, Nat. Rev. Immunol . 13 (4): 227-242, 2013).

As used herein, the term " chemokine " refers to a chemotactic cytokine that modulates cell migration and location by activating a G protein-linked chemokine receptor (GPCR) that includes a 7-transmembrane portion. Chemokines are divided into four subfamilies, CC, CXC, CX3C and XC, depending on the location of the first two N-terminal cysteine residues. In particular, in a tumor microenvironment (hereinafter referred to as 'TME') composed of cells of the host surrounding the tumor and the tumor, tumor-associated host cells and cancer cells secrete various chemokines, Various types of cells that mediate the balance between tumor and tumor-promoting responses are replenished and activated. Furthermore, besides the role of chemotaxis, chemokines also participate in other tumor-related processes, including tumor cell growth, neovascularization and metastasis. Therefore, a polynucleotide encoding a chemokine receptor that is not expressed in NK101 of the present invention can be transduced and expressed by increasing the specific mobility of NK101 cells of the present invention against cancer cells (Yang et al ., J. Immunother Cancer , 3 (Suppl 2): P24, 2015).

In the recombinant NK cell, the chemokine receptor may be CCR or CXCR, and the CCR may be CCR1, CCR2, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CXCR6 or CXCR7.

As used herein, the term "apoptosis inducing ligand" refers to a protein that binds to a receptor on the surface of a cell and induces apoptosis of the target cell. Representative examples of such apoptosis inducing ligands include TRAIL (TNF-related apoptosis-inducing ligand) and FasL (Fas ligand).

In the recombinant NK cells, the apoptosis inducing ligand may be TRAIL or FasL.

As used herein, "operably linked to" means that a particular polynucleotide is linked to another polynucleotide so that it can perform its function. In other words, the fact that a polynucleotide encoding a specific protein is operatively linked to a promoter implies that it is transcribed into mRNA by the action of the promoter and is linked so as to be translated into the protein, and a polynucleotide encoding a specific protein The fact that the particular protein is operably linked to a polynucleotide encoding another protein can be expressed in the form of another protein and a fusion protein.

Modulators that enable expression in the eukaryotic and prokaryotic cells are well known to those skilled in the art. As discussed above, these usually include regulatory factors responsible for transcription initiation and, optionally, poly-A signals responsible for transcription termination and stabilization of transcripts. Additional regulatory factors may include translation enhancing factors and / or natural-combining or heterologous promoter regions in addition to transcriptional regulatory factors. For example, possible regulatory elements enabling expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, the RSV (low-grade sarcoma virus) promoter, the human renal element 1 alpha-promoter, the glucocorticoid-inducible MMTV-promoter (Moloney murine tumor virus), a metallothionein-inducible or tetracycline-inducible promoter, or an amplifying agent such as a CMV or SV40-amplification agent. For neuronal expression, it is contemplated that neurofilament-promoter, PGDF-promoter, NSE-promoter, PrP-promoter or thy-1-promoter may be used. Such promoters are known in the art and described in the literature (Charron, J. Biol. Chem. 1995, 270: 25739-25745). For prokaryotic expression, a number of promoters have been disclosed, including lac-promoter, tac-promoter or trp promoter. In addition to factors capable of initiating transcription, the regulatory elements include a transcription termination signal such as an SV40-poly-A site or a TK-poly-A site downstream of the polynucleotide according to an embodiment of the invention You may. In this document, suitable expression vectors are known in the art, including Okayama-Berg cDNA expression vectors pcDV1 (Parmacia), pRc / CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL , pX (Pagano (1992) Science 255, 1144-1147), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris et al ., Cell 75, 791-803, 1995) Expression vectors, such as lambda gt11 or pGEX (Amersham-Pharmacia). In addition to the nucleic acid molecules of the present invention, the vector may further comprise a polynucleotide encoding a secretion signal. Such secretion signals are well known to those skilled in the art. And, depending on the expression system used, a leader sequence capable of directing the peptides of the present invention to the cell compartment is combined with the coding sequence of the polynucleotide according to one embodiment of the present invention, Or a leader sequence capable of direct secretion of its protein into the cytoplasmic or extracellular medium. Alternatively, the heterologous sequence can encode a fusion protein comprising a C-terminal or N-terminal tag peptide which confers desired properties such as stabilization or simple purification of the expressed recombinant product. These tags include but are not limited to FLAG, GST (glutathione S transferase), HisX6, and the like. When a vector according to one embodiment of the present invention is transfected into a suitable host cell or non-human host host, the host cell or host host is maintained under conditions suitable for high level expression of the nucleotide sequence.

 As used herein, the term "genetic recombinant or genetic engineering" is intended to encompass all types of polynucleotides, including, but not limited to, poly Means that the host cell or host entity contains a nucleotide or vector other than its own genome. In addition, a polynucleotide or vector according to one embodiment of the present invention may be present in a genetically modified host cell or host entity as an independent molecule outside the genome, preferably as a replicable molecule, or may be a host cell or host entity Lt; RTI ID = 0.0 > genomic < / RTI >

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating and preventing cancer, which contains the isolated NK cell and / or the recombinant NK cell line as an active ingredient.

In the above pharmaceutical composition, the cancer may be selected from the group consisting of lung cancer, stomach cancer, liver cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colon cancer, Cancer of the uterus, cancer of the urethra, cancer of the urethra, cancer of the urethra, cancer of the prostate, prostate cancer, childhood solid tumor, bladder cancer, kidneys, kidney cancer, endometrial carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma Cancer, kidney cell carcinoma, renal pelvic carcinoma, cholesteatoma tumor, glioma, or pituitary adenoma, and the like.

In the above pharmaceutical composition, the cancer may be a metastatic cancer.

The composition of the present invention may contain one or more known active ingredients having an anticancer effect together with the isolated NK cells and / or recombinant NK cells.

The composition may additionally contain a pharmaceutically acceptable excipient or diluent in addition to the carrier.

The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the composition according to one embodiment of the present invention may be formulated using methods known in the art to allow rapid release, or sustained or delayed release, of the active ingredient upon administration to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

Compositions according to one embodiment of the present invention may be administered in a variety of routes including, for example, oral, parenteral, e.g., suppository, transdermal, intravenous, intraperitoneal, intramuscular, And may also be administered using an implantable device for sustained or continuous or repeated release. The number of administrations can be administered once or several times a day within a desired range, and the administration period is not particularly limited.

The administration route of the composition according to one embodiment of the present invention may be administered through any conventional route as long as it can reach the target tissue. Such an administration route may be, but not limited to, parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, and intrathecal administration.

The composition according to one embodiment of the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, aqueous carriers for parenteral administration such as aqueous glucose and glycols, etc., and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention may contain various additives such as a suspending agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, an interface activator, a diluent, an excipient, a pH adjuster, An antioxidant, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington ' s Pharmaceutical Sciences, Current Edition.

The dosage for the patient of the pharmaceutical composition depends on many factors, including the patient's height, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently . Usually, NK cells for cell therapy are administered at a dose of about 10 ⁹ to 10 ¹⁰ cells per m ^{2 of} body surface area. Thus, it is appropriate that about 2 x 10 < 10 ^> cells are administered on a standard adult (about 60 kg) basis, but the dose will vary depending on the various conditions of the patient and the type and amount of the drug have. Accordingly, the isolated NK cells or recombinant NK cells of the present invention which are pharmaceutically active may be administered in an amount of 10 ⁶ to 10 ¹⁰ cells / kg (body weight), and administration below or above the above example range, particularly, ≪ / RTI > If the administration method is continuous infusion, it should be within the range of 10 ³ to 10 ⁹ cells per 1 kg of body weight per minute.

According to another aspect of the present invention there is provided a method of treating said cancer-bearing organism comprising administering a therapeutically effective amount of said isolated NK cells and / or recombinant NK cells to a subject having cancer .

As used herein, the term "therapeutically effective amount" means an amount that significantly inhibits the death of cancer cells or at least the growth of cancerous tissue.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. And is provided to fully disclose the scope of the invention to a person having ordinary skill in the art.

Example 1: Preparation of NK cell line of the present invention

To prepare NK cell-derived cell lines, the following procedures were performed. Patient-derived extranodal NK lymphocytic carcinoma was placed on a 40-μm strainer and infused with Cellgro ^® (Sigma) containing 20% fetal bovine serum (GE healthcare, Pittsburgh, USA) and 1% antibiotic (Life technologies, Maryland, USA) 10 ml of stem cell growth medium (SCGM; CellGenix, Freiburg, Germany, hereinafter referred to as 'NK media') was added, and the cells were detached into single cells using the shear force of a 5 ml syringe piston. NK cells in a single cell suspension were separated using an NK separation kit (Milltenyi Biotec, Bergisch Gladbach, Germany) and then added with 1000 U / ml of human recombinant IL-2 (rhIL-2; Prometheus, San Diego, USA) NK media for 3 weeks. NK media containing rhIL-2 was added twice a week during culture, and a stable cell line was formed by continuing cultivation of the mitotic cell line to 30 passages (FIG. 1A). It was confirmed by microscopic observation that the cell line had a characteristic of forming a spheroid in culture (Fig. 1B), and that the cell proliferation was dependent on IL-2 (Fig. 1C). In addition, it was confirmed that CD56 was expressed without expressing CD3, CD20, and CD16, and that the origin of the cell was NK cell (Fig. 1d), and that the cell division ability was IL-2 dependent (Fig. 1e) . The NK101 cell line was identified by confirming the characteristics of cell coin and NK cells. The cell line was designated as 'NK101 cell line' and the Korean Collection for Type Culture (KCTC) was constructed at Korea Institute of Bioscience and Biotechnology, On August 7, 2017, and was granted the accession number of KCTC 13305BP on August 24, 2017. The depositary is an international depository institution under the Treaty of Buttepe.

Example 2: Culture conditions and cell division ability of the NK cell line of the present invention

In order to establish the culture conditions for the NK101 cell line prepared in Example 1, 500 IU / ml rhIL-2 was added to SCGM, IMDM, MEMα and RPMI1640 culture medium containing 20% fetal bovine serum to compare the growth. As a result, as shown in FIG. 2A, the NK101 cells of the present invention maintained the cumulative proliferative capacity (CPDL) and cell survival rate well in SCGM, IMDM, and MEMa, but the cell survival rate over time in RPMI- A phenomenon of remarkably falling was observed. These results suggest that alternative culture media (such as IMDM and MEMa), which are not expensive SCGMs, can lower cell line production costs. As a result of comparing cell division ability with NK-92 cell line, established NK cell line, it was confirmed that the amount of NK101 cells ultimately obtained during the 20-day subculture was about 100 times that of NK-92 (b) . This suggests that the productivity of the NK101 cells of the present invention is superior to those of the conventional NK-92 cells, and that the NK101 cells of the present invention are very advantageous in terms of economy.

Comprehensive characteristics of the NK101 cells of the present invention are summarized in Table 2 below.

Main features of NK101 cells of the present invention Item NK101 cells Clinical data Age / Sex 56 year old man race Asian Diagnosis Extranodal NK / T lymphoma (extranodal NK / T lymphoma) Treatment status At diagnosis  Establishment year 2016 Cell culture Growth aspect In suspension, multiple cell aggregates Doubling Time 18-32 hours Maximal cell concentration 1.2 x 10 < ⁶ > cells / ml Minimal cell concentration 1 x 10 < ⁵ > cells / ml Cytokine dependence IL-2 dependent (500 IU / ml) Optimal disruption Every 2-3 days Immunological characteristics
T / NK marker CD2 ⁺ , CD3 ^- , CD4 ^- , CD7 ^- , CD8 ^- , CD16 ^- , CD56 ⁺ B cell marker CD10 ^- , CD19 ^- , CD20 ^- Bone marrow mononuclear marker CD13 ^- , CD14 ^- , CD33 ⁺ NK cell activation receptor NKp46 ⁺ , NKp44 ^- , NKp30 ⁺ , NKG2D ⁺ , NKG2C ^- NK cell inhibitory agent KIR2DL1 ^- , KIR2DL2 ^- , ILT2 ^- Offspring / activation marker ^{CD34 -, HLA-DR +,} FAS + Attachment marker CD11a ⁺ , CD18 ⁺ , CD54 ⁺ Functional characteristic NK activity Intermediate NK activity Cytokine production Production of significant IFN-γ in the presence of IL-2 Cytokine receptor CD25 ⁺ , CD122 ⁺ , CD132 ⁺ , CD127 ^- Chemokine receptor CCR4 ⁺ , CCR6 ⁺ , CCR7 ⁺ , CCR8 ⁺ , CXCR3 ⁺ , CXCR4 ⁺

Example 3: Identification of the NK cell line phenotype of the present invention

The NK101 cell line of the present invention prepared in Example 1 had the characteristics of floating cells and the expression of the surface antigen of the cells was confirmed by flow cytometry. In the case of the T / NK cell marker, the NK101 cell line of the present invention expresses CD2, CD56, which is surface antigens of NK cells, but does not express CD16, and expresses the surface antigens CD3, CD4, CD8, TCRαβ and TCRγδ (Fig. 3). ≪ / RTI > In addition, surface antigen (CD10, CD19, CD20), granulocyte-expressing antigen (CD13, CD23), dendritic and mononuclear cell surface antigens (CD1a, CD11c, CD14) , And was found to express CD33 and HLA-DR, surface antigens of myeloid cells (Fig. 4). In addition, the NK101 cell line of the present invention expressed NKp30, NKp46 and NKG2D, which are activating receptors of NK cells (Fig. 5A), and ILT2, KIR2DL1 and KIR2DL2 / DL3 which are inactivated receptors were not expressed, but CD94 and CD159a (Fig. 5B). CD18, CD54, DNAM-1, 2B4 and the like acting as co-receptors for T cell adhesion and NK cell activation (FIG. 5C). In addition, the NK101 cell line of the present invention showed high expression of perforin and IFN-y, which are involved in cytotoxicity and immunological activation of NK cells, while low expression of TRAIL (FIG. 6), and among cytokine receptors of NK cells IL-2 receptor (CD122, CD132) and CD127 (IL-7 receptor) were not expressed (FIG. 7). The role of IFN-y is typically that it induces strong anticancer activity when NK cell activation, macrophage activation, induction of homologous IgG antibody conversion, Th2 inhibition, and MHC expression increase, and high INF-γ activity. The NK101 cell line of the present invention is distinguished from other NK cell lines in that CD25 is highly expressed, which is an indicator of activated NK cells and is known as a marker of NK cells having particularly high fission ability (Clausen, J. et al. , Immunobiology , 207 (2): 85-93, 2003), which is well suited for mass production of cell therapeutic agents. CCR4, CCR6, CCR7, CXCR3, and CXCR4 were expressed among the chemokine receptors related to the mobility of NK cell line, but other CCRs and CXCRs were not expressed (FIGS. 8A and 8B). In conclusion, the NK101 cell line of the present invention expresses CD2, CD11a, CD26, CD45, CD54, CD56 and HLA-DR, and does not express CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, And had a phenotype of activated NK cells (Figs. 3, 4 and 5c). The NK101 cell line of the present invention is distinguishable from NK-92 cells in that it is HLA-DR positive, unlike NK-92 which is HLA-DR negative. In addition, the NK101 cell line of the present invention expresses NKp30, NKp46, low levels of NKG2D, CD94 and KRLB-1 known as NK cell activation receptors (Fig. 5A) and expresses inactivating receptors ILT2, KIR2DL1 and KIR2DL2 / (Fig. 5B). It was confirmed that perforin, IFN-y and TRAIL, which are involved in cytotoxicity and immunological activation of NK cells, are expressed (Fig. 6). Among the cytokine receptors of NK cells, CD25, which expresses the IL-2 highly positive receptor, and CD127, which is a receptor of IL-7, were not expressed (FIG. 7). Among the chemokine receptors related to the mobility of NK cell lines, CCR4, CXCR3, and CXCR4 were expressed, while other types of CCR and CXCR were not expressed (FIGS. 8A and 8B). The NK101 cell line of the present invention is differentiated from other NK cell lines in that CD25 is highly expressed. CD25 is an indicator of activated NK cells and is known as a marker of NK cells having particularly high fission ability (Clausen, J. et al ., Immunobiology , 207 (2): 85-93, 2013), it can be seen that the NK101 cell line of the present invention is a cell line highly suitable for mass production of a cell therapeutic agent.

Expression of various cell markers of NK101 cells of the present invention is summarized in Table 3 below.

The marker characteristics of the NK101 cells of the present invention antigen Expression antigen Expression System marker Functional marker CD1a - CD95 (FAS) +++ CD2 +++ CD178 (FAS-L) + CD3 - CD107a +++ CD4 - TRAIL (CD253) + CD5 - Perforine +++ CD8 - Granzyme B +++ CD10 - IFN? +++ CD11a +++ Chemokine receptor CD11c - CCR1 + CD13 + CCR2 - CD14 + CCR3 - CD16 - CCR4 +++ CD18 +++ CCR5 + CD19 - CCR6 +++ CD23 - CCR7 +++ CD33 +++ CCR8 ++ CD45 +++ CCR9 + CD56 +++ CXCR1 - CD57 - CXCR2 - CD161 ++ CXCR3 +++ Activated receptor CXCR4 +++ 2B4 +++ CXCR5 - NKp30 ++ CXCR6 - NKp44 - CXCR7 - NKp46 +++ Cytokine receptor NKp80 - CD25 (IL-2Ra) +++ NKG2C - CD122 (IL-2Rb) ++ NKG2D ++ CD132 (common gamma chain) +++ Inhibitory receptor CD127 (IL-7Ra) - CD85j (ILT2) - Other markers CD94 +++ TCRαβ - CD158 (KIR2DL1 / S1 / S3 / S5) - TCRγδ - CD158b (KIR2DL2 / DL3) - HLA-DR +++ CD159a +++ -, voice;
+, ≪ 10% positive;
++, 10-69% positive;
+++, 70-100% positive (% indicates the proportion of positive cells in the cell population) Adherent molecule DNAM-1 (CD226) +++ ICAM-1 (CD54) +++ CD62L ++

Example 4: Preparation of NK101 in vitro  And in vivo  Cytotoxicity check

The following experiment was conducted to confirm the cancer cell killing ability of the NK101 cell line prepared in Example 1 and identified as a phenotype. Specifically, human-derived cancer cells NCI-H460 (lung cancer), U373 (brain cancer), A2780 (ovarian cancer), HCT116 (colorectal cancer), SK-BR3 (breast cancer), THP-1 (Acute myelogenous leukemia) and K562 (chronic myelogenous leukemia) were inoculated in a 24-well culture dish at a concentration of 3 x 10 ⁴ cells / ml. Since then NK101 Cells and controls were suspended in 1 ml medium with various effector cell-to-target cell ratio (E: T ratio = 1: 1, 2: 1, and 4: 1) and then incubated with the cancer cells for 24 hours. After culturing, all cells were collected and the cells were recovered from each well and centrifuged, and the cell pellet was suspended in FACS buffer. Was suspended in a 100 ㎕ diluted again centrifuged to pellet the cells formed 1 ㎕ LIVE / DEAD ^ㄾ Fixable Near-IR Dead Stain Kit ( Life Technologies) FACS buffer and then reacted at 4 ℃ 20 minutes. After washing twice with FACS buffer, the cell pellet was suspended in 100 μl of 1X Annexin V binding buffer in a solution diluted with 5 μl of Annexin V APC (Biolegend, USA), and reacted at room temperature for 20 minutes. (Annexin V-negative / LIVE / DEAD- negative), early apoptotic cells (annexin V-positive / LIVE / DEAD-negative), and late apoptotic cells (annexin V- LIVE / DEAD-positive) and necrotic cells (annexin V-negative / LIVE / DEAD-positive). As shown in FIG. 9A, the NK101-treated group shows cell killing ability against various human cancer cell lines.

In addition, THP-1 cells were used to compare the cytotoxicity of NK101 and NK-92 cell lines with those of THP-1 cells (FIG. 9b). In addition, when IFN gamma in the co-culture solution of THP-1, NK101 and NK-92 was confirmed by ELISA, it was confirmed that INFγ expressed in NK101 cell line was significantly higher than that expressed in NK-92 (FIG. Based on the above facts, it has been found that NK101 cells have an advantage in that they have an excellent immunoreactive cytokine secretion ability such as IFNγ even though they have cancer cell killability similar to that of NK-92 cells, .

In order to confirm the anticancer effect of NK101 cells in vivo , the present inventors administered 10 ⁶ THP-1 cell lines expressing luciferase to immunodeficient mice (NSG mice) After 3 days of cancer treatment, 5 × 10 ⁶ NK101 cells or NK-92 cells were administered four times at intervals of 2 days, and 15 mg / kg of luciferin was intraperitoneally administered to IVIS spectrum in vivo imaging system by imaging through (Perkin Elmer, USA) confirmed the therapeutic effect of these NK cells (Fig. 9d). As a result, both NK101 and NK-92 cells showed similar anti-cancer effects.

Example 5: Preparation of transgenic NK101 cells expressing apoptosis-inducing ligand TRAIL and evaluation of antitumor activity

(TRAIL) (GenBank No. AAH32722.1) DNA cassette (Fig. 10A) was first inserted into pBD2.5, a transfer vector for lentivirus production, to construct a cell line expressing the TRAIL gene, which is an apoptosis inducing ligand, / TRAIL vector.

For the production of lentivirus, the 12 占 퐂 pBD2.5 / TRAIL plasmid, 12 占 퐂 packaging psPAX2 plasmid and 2.4 占 퐂 pMD.G plasmid were ligated with lipofectamine 2000 (Lipofectamine 2000, Invitrogen, USA) (Invitrogen, Carlsbad, Calif., USA). After incubation for about 48-72 hours, the virus was harvested and mixed with a lentivirus concentrator (Lenti X Concentrator, Clontech Laboratories, USA). After overnight incubation at 4 ° C, viruses were collected by centrifugation at 4,000 RPM and re-released into culture.

To prepare TRAIL-expressing NK cell line, the prepared lentivirus and protamine sulfate were reacted at 37 ° C for 4 hours to infect NK101 cell line. After a total of 2 infections, TRAIL expression was confirmed by flow cytometry at 72 hours after infection, and one week later, only NK101 cells expressing TRAIL were selectively isolated using a fluorescence activated cell sorter .

To confirm whether the NK101 cell line expressing TRAIL enhances the effect on cancer cell death in vitro , cancer cells and NK101-TRAIL cells were co-cultured to produce apoptotic cells and dead cells in Example 3 Were analyzed according to the method used. The target cancer cells are HCT116 human colon cancer cell lines. For comparison, NK101 cell line which does not express the transgene was used as a control.

As a result, as shown in FIG. 10B, it was confirmed that NK101 cells expressing TRAIL kill cancer cells at an E: T ratio of about 1: 1 to about 60% and 3: 1 to about 75%. Overall, NK101 cells are significantly increased in anticancer effects due to the introduction of apoptosis inducing ligands such as the TNF family.

Example 6: Preparation of transformed NK101 cells expressing CD7 and CD28, which are NK cell assistive factors, and evaluation of antitumor activity

(SEQ ID NO: 1) encoding the P2A sequence so that CD7 (GenBank No. AAH13297.1) and CD28 (GenBank No. AAH93698.1), which are NK cell assisted stimulatory factors that are not expressed in the NK101 cell line of the present invention, (FIG. 11A) was constructed, inserted into pBD2.5, a transfer vector for lentivirus production, and constructed as a pBD2.5 / CD7-CD28 vector. , And then introduced into NK101 cell line to isolate NK101 / CD7-CD28 transformed cell line.

In order to confirm the increase of cancer cell killing effect by CD7 and CD28, NK cell-assisted stimulating factors, NK101 / CD7-CD28 cell line and cancer cells were co-cultured in vitro at 1: 1 ratio. The target cancer cells used herein were JEKo-1 expressing the CD7 ligand, CD80 which is a ligand of CD28, U937 expressing CD86, KG-1 and THP-1 expressing SECTM-1 and NK101 was used as a control cell Respectively.

As a result, as shown in FIGS. 11B and 11C, the cancer cell-specific killing ability of NK101 / CD7-CD28 was 30% (Jeko-1), 56.6% (U937), 41.3% (KG-1) -1)%, which is about 1.6 times that of NK101 used as a control group compared to 15% (Jeko-1), 35.9% (U937), 24.9% (KG-1) and 23% (THP-1) It is confirmed that it increases up to 2 times.

Example 7: Inhibition of cell proliferation and anticancer efficacy according to irradiation

The NK101 cell of the present invention is a cell line derived from human cancer, and when used as a therapeutic agent, it may be able to multiply in the body, which may cause safety problems. Thus, the proliferation of NK101 cells of the present invention was observed by irradiation of 1, 5, 10, and 20 Gy, respectively (FIG. 12A). As a result, the proliferation of NK101 cell line was inhibited by irradiation of 5 Gy or more Respectively. In addition, when the cytotoxicity of NK101 cell line after radiation irradiation was observed by 4: 1 co-culture with HCT116 cells, it was confirmed that the cytotoxicity of NK101 did not significantly decrease upon irradiation of about 5 Gy or less (FIG. Comprehensively, it can be seen that NK101 cells can maintain cancer cell killing ability without human proliferation when human body is administered through the irradiation method. Therefore, the NK101 cell of the present invention is expected to be used as a cell therapy agent for more efficient and economical cancer treatment.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation example  1: Preparation of injection

Isolated NK101 cells or recombinant NK101 cells at 1 × 10 ⁶ to 5 × 10 ¹⁰ cells

pH adjuster

Stabilizer <

Use sterilized distilled water up to 100%

(2 ml) per ampoule according to the usual injection preparation method.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> Korea Institute of Science and Technology <120> A novel recombinant exosome and use thereof <130> PD17-5513 <150> KR 2016-0090232 <151> 2016-07-15 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1536 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding VSV-G protein <400> 1 atgaagtgcc ttttgtactt agccttttta ttcattgggg tgaattgcaa gttcaccata 60 gtttttccac acaaccaaaa aggaaactgg aaaaatgttc cttctaatta ccattattgc 120 ccgtcaagct cagatttaaa ttggcataat gacttaatag gcacagcctt acaagtcaaa 180 atgcccaaga gtcacaaggc tattcaagca gacggttgga tgtgtcatgc ttccaaatgg 240 gtcactactt gtgatttccg ctggtatgga ccgaagtata taacacattc catccgatcc 300 ttcactccat ctgtagaaca atgcaaggaa agcattgaac aaacgaaaca aggaacttgg 360 ctgaatccag gcttccctcc tcaaagttgt ggatatgcaa ctgtgacgga tgccgaagca 420 gtgattgtcc aggtgactcc tcaccatgtg ctggttgatg aatacacagg agaatgggtt 480 gattcacagt tcatcaacgg aaaatgcagc aattacatat gccccactgt ccataactct 540 acaacctggc attctgacta taaggtcaaa gggctatgtg attctaacct catttccatg 600 gacatcacct tcttctcaga ggacggagag ctatcatccc tgggaaagga gggcacaggg 660 ttcagaagta actactttgc ttatgaaact ggaggcaagg cctgcaaaat gcaatactgc 720 aagcattggg gagtcagact cccatcaggt gtctggttcg agatggctga taaggatctc 780 tttgctgcag ccagattccc tgaatgccca gaagggtcaa gtatctctgc tccatctcag 840 acctcagtgg atgtaagtct aattcaggac gttgagagga tcttggatta ttccctctgc 900 caagaaacct ggagcaaaat cagagcgggt cttccaatct ctccagtgga tctcagctat 960 cttgctccta aaaacccagg aaccggtcct gctttcacca taatcaatgg taccctaaaa 1020 tactttgaga ccagatacat cagagtcgat attgctgctc caatcctctc aagaatggtc 1080 ggaatgatca gtggaactac cacagaaagg gaactgtggg atgactgggc accatatgaa 1140 gacgtggaaa ttggacccaa tggagttctg aggaccagtt caggatataa gtttccttta 1200 tacatgattg gacatggtat gttggactcc gatcttcatc ttagctcaaa ggctcaggtg 1260 ttcgaacatc ctcacattca agacgctgct tcgcaacttc ctgatgatga gagtttattt 1320 tttggtgata ctgggctatc caaaaatcca atcgagcttg tagaaggttg gttcagtagt 1380 tggaaaagct ctattgcctc ttttttcttt atcatagggt taatcattgg actattcttg 1440 gttctccgag ttggtatcca tctttgcatt aaattaaagc acaccaagaa aagacagatt 1500 tatagaca tagagatgaa ccgacttgga aagtaa 1536

Claims (15)

Isolated human NK cells with the following characteristics:
CD2, CD11a, CD25, CD45, CD54, CD56 and HLA-DR are positive; And
CD1a, CD3, CD4, CD8, CD14, CD16, CD20, CD23, CD34, TCRαβ and TCRγδ are negative. The method according to claim 1,
Isolated NK cells further comprising the following features:
CD18, CD33, CD122, CD132, CD159 and FAS are positive;
High expression of INF gamma upon IL-2 treatment; And
CD7, CD10, CD11c, CD13, CD19, CD127, CD158a (KIR2DL1), CD158b (KIR2DL2), NKG2C, and ILT2 are negative. 3. The method of claim 2,
Isolated NK cells further comprising the following features:
CD94, DNAM1, 2B4, NKp30, NKp46, and NKG2D;
NKp44, NKp80, KIR2DL1 and KIR2DL2 / DL3;
CCR4, CCR6, CCR7, CCR8, CXCR3, and CXCR4 are expressed; And
CCR1, CCR2, CCR3, CCR5, CCR9, CXCR1, CXCR2, CXCR5, CXCR6 and CXCR7 are not expressed. The method according to claim 1,
Isolated NK cells deposited with Accession No. KCTC 13305BP. A method for producing an NK cell comprising the steps of: transfecting an isolated NK cell of any one of claims 1 to 4 with a gene construct operably linked to a promoter, the polynucleotide encoding one or more foreign proteins, Recombinant NK cells. 6. The method of claim 5,
The exogenous protein may be a cancer cell specific receptor for cancer cell targeting or a protein specifically binding to the ligand, an immunomodulatory polypeptide comprising a costimulation domain, a chemokine receptor or an apoptosis-inducing ligand, , Recombinant NK cells. The method according to claim 6,
Wherein said immunomodulatory polypeptide is selected from the group consisting of CD28, ICOS, CTLA4, PD1, BTLA, DR3, 4-1BB, CD2, CD40, CD30, CD27, SLAM, 2B4, NKG2D) / DAP12, TIM1, TIM2, TIM3, TIGIT, CD226, , LAG3, B7-1, B7-H1, GITR, HVEM or OX40L or their complementary stimulatory domains. The method according to claim 6,
Wherein said chemokine receptor is CCR (CC chemokine receptor) or CXCR (CXC chemokine receptor). 9. The method of claim 8,
Wherein said CCR is CCR1, CCR2, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 or CCR10. 9. The method of claim 8,
Wherein said CXCR is CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CXCR6 or CXCR7. The method according to claim 6,
Wherein said apoptosis inducing ligand is TRAIL or FasL. A pharmaceutical composition for treating cancer comprising the isolated NK cell of any one of claims 1 to 4 as an active ingredient. A pharmaceutical composition for treating cancer comprising the recombinant NK cell of claim 5 as an active ingredient. A method of treating cancer in an individual comprising administering a therapeutically effective amount of an isolated NK cell of any one of claims 1 to 4 to a subject having cancer. A method for treating cancer in a subject comprising administering a therapeutically effective amount of the recombinant NK cell of claim 5 to a subject having cancer.

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