Natural killer cells generated from induced pluripotent stem cells, methods for generating the same, and uses thereof

Abstract

The present invention relates to natural killer cells generated and expanded in large quantities from induced pluripotent stem cells, and pharmaceutical compositions containing said natural killer cells as active ingredients. The natural killer cells according to the present invention showed tumor lytic activity at the cell and animal levels. Furthermore, the expression of genes and cytokines involved in tumor death and immune cell activity was increased compared to natural killer cells isolated and activated from blood. It was also confirmed that the method for inducing differentiation of natural killer cells derived from induced pluripotent stem cells according to the present invention is a method for generating a large number of cells within a short period of time by using a simple method compared to conventional induction methods using induced pluripotent stem cells. Therefore, it is believed that the method for differentiating natural killer cells derived from induced pluripotent stem cells according to the present invention, and the natural killer cells generated by said method, can be used to treat various diseases, including cancer.

Description

[Technical field]

[0001] The present invention relates to natural killer cells generated from induced pluripotent stem cells, methods for generating the natural killer cells, and uses thereof. [Background technology]

[0002] Natural killer cells (NK cells) are one of the cytotoxic lymphocytes important for innate immunity. Natural killer cells have cytotoxicity that can directly kill not only virus-infected cells but also cancer cells. In general, immune cells detect major histocompatibility complexes (MHC) displayed on the surface of infected cells, induce cytokine release, and induce the death of infected cells by cell lysis or cell apoptosis.

[0003] On the other hand, natural killer cells can recognize and eliminate abnormal cells such as cancer cells without antibodies or MHC, so natural killer cells can induce an immediate immune response. Natural killer cells are known to attack cancer cells, prevent the development, proliferation, and metastasis of cancer cells, and effectively control cancer stem cells, which play an important role in cancer recurrence. Therefore, various studies have been attempted to treat cancer using natural killer cells. Therefore, techniques for mass production of allogeneic and autologous human natural killer cells have also emerged as important techniques.

[0004] The main source of human natural killer cell resources is the method of isolating and expanding natural killer cells from blood. However, natural killer cells account for only about 10% of lymphocytes in blood. In order to apply natural killer cells to various fields including anti-cancer therapy, it is necessary to mass-produce natural killer cells in vitro with high yields, but this is not yet at a level that can be applied clinically (Korean Patent Application Publication No. 10-2018-0117829). Recently, technology to produce natural killer cells with excellent differentiation ability through differentiation induction culture from stem cells such as human hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells has also been actively researched. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have endeavored to develop a method for producing human natural killer cells with high efficiency. As a result, natural killer cells were produced from isolated induced pluripotent stem cells (iPSCs), and the produced natural killer cells were expanded in large quantities and their characteristics were analyzed. As a result, it was confirmed that the natural killer cells exhibit characteristics that are distinct from existing blood-derived natural killer cells. Based on the above, the present inventors have completed the present invention. [Means for solving the problem]

[0006] In order to achieve the above-mentioned object, in one aspect of the present invention, there are provided natural killer cells expressing any one protein selected from the group consisting of CD25, CD9, CD80, and combinations thereof; a pharmaceutical composition comprising the aforementioned natural killer cells as an active ingredient; and a method for producing natural killer cells.

[0007] In another aspect of the invention there is provided the use of natural killer cells for the prevention or treatment of disease.

[0008] In another aspect of the invention, a method for preventing or treating a disease is provided comprising administering natural killer cells. Effect of the Invention

[0009] The natural killer cells according to the present invention showed tumor lytic activity at the cell and animal levels. Furthermore, the expression of genes and cytokines involved in tumor death and immune cell activity was increased compared to natural killer cells isolated and activated from blood. Furthermore, the method for differentiating natural killer cells from iPSCs according to the present invention can generate a large number of cells within a short period of time compared to conventional methods for differentiating natural killer cells from iPSCs. Furthermore, the activity of the natural killer cells generated by this method was confirmed to be excellent. Therefore, the method of the present invention for differentiating natural killer cells from iPSCs and the natural killer cells generated by this method can be used to treat various diseases including cancer. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of differentiation of natural killer cells from induced pluripotent stem cells (iPSCs) in the presence of autologous feeder cells. [Diagram 2] FIG. 2 outlines a method for differentiating natural killer cells from induced pluripotent stem cells (iPSCs) in the presence of autologous feeder cells. [Diagram 3] FIG. 3 shows the results obtained by confirming morphological changes at each stage of differentiation of iPSCs into NK cells using an optical microscope. [Figure 4] FIG. 4 shows the results obtained by photographing cell culture vessels in groups in which autologous feeder cells were maintained and groups in which autologous feeder cells were removed after the hematopoietic stem cell differentiation stage during differentiation of iPSCs into NK cells. [Diagram 5]FIG. 5 shows the results obtained by observing using an optical microscope the difference between the group in which autologous feeder cells were maintained and the group in which the autologous feeder cells were removed after the hematopoietic stem cell differentiation step during differentiation of iPSCs into NK cells. [Figure 6] FIG. 6 is a graph showing the results obtained by analyzing the number of natural killer cells (suspension cells) generated at each differentiation step during differentiation of iPSCs into NK cells in a group in which autologous feeder cells were maintained after the hematopoietic stem cell differentiation step and in a group in which autologous feeder cells were removed. [Figure 7] FIG. 7 shows the results obtained by flow cytometric analysis of key natural killer cell markers in cells differentiating into natural killer cells in groups in which autologous feeder cells were maintained and in which autologous feeder cells were removed after the hematopoietic stem cell differentiation stage during differentiation of iPSCs into NK cells. [Figure 8a] 8a-8c are diagrams and graphs showing the results obtained when differentiating iPSCs into natural killer cells, ascertaining the number of iPSC colonies attached according to Condition 1-Condition 6, respectively (8a), the total number of cells / attached vessel area (cm2) for each condition (8b), and the total number of iPSC colonies per T-150 flask for each condition (8c). [Figure 8b] 8a-8c are diagrams and graphs showing the results obtained when differentiating iPSCs into natural killer cells, ascertaining the number of iPSC colonies attached according to Condition 1-Condition 6, respectively (8a), the total number of cells / attached vessel area (cm2) for each condition (8b), and the total number of iPSC colonies per T-150 flask for each condition (8c). [Figure 8c]8a-8c are diagrams and graphs showing the results obtained when differentiating iPSCs into natural killer cells, ascertaining the number of iPSC colonies attached according to Condition 1-Condition 6, respectively (8a), the total number of cells / attached vessel area (cm2) for each condition (8b), and the total number of iPSC colonies per T-150 flask for each condition (8c). [Figure 9] FIG. 9 shows the results obtained by confirming, using an optical microscope, the morphological changes of each cell differentiated according to stages under conditions 1 to 6 when iPSCs were differentiated into NK cells. [Figure 10] FIG. 10 is a graph showing the results obtained by confirming the amount of differentiated natural killer cells (non-adherent cells) under each condition. [Figure 11] FIG. 11 is a graph showing the results obtained by analyzing the expression rates of cell surface proteins (markers) of natural killer cells differentiated under conditions 1 to 6 when iPSCs were differentiated into NK cells. [Figure 12] FIG. 12 is a set of diagrams and graphs showing the results obtained by analyzing the number of induced pluripotent stem cell colonies that adhered when iPSCs were differentiated into NK cells under each of conditions 1 to 6. [Figure 13] FIG. 13 shows the results obtained by observing, using an optical microscope, morphological changes in differentiated cells at each stage under each condition (A to D) when iPSCs were differentiated into NK cells. [Figure 14] Figure 14 is a graph showing the results obtained by differentiating iPSCs into NK cells and confirming the expression rate of cell surface marker proteins (markers) of natural killer cells differentiated under each condition (A to D) by flow cytometry. [Figure 15] FIG. 15 shows the results of flow cytometry analysis of cells at the mesoderm cell stage (day 4 of differentiation) during differentiation from induced pluripotent stem cell colonies (iPSC colonies) into NK cells. [Figure 16]FIG. 16 shows the results of flow cytometry analysis of cells differentiated into hematopoietic stem cells through the mesodermal cell stage during differentiation of iPSC colonies into NK cells (day 12 of differentiation). [Figure 17a] 17a and 17b show the results of flow cytometry analysis of cells differentiated from iPSC colonies through the mesoderm and hematopoietic stem cell stages to NK cells (day 26 of differentiation). [Figure 17b] 17a and 17b show the results of flow cytometry analysis of cells differentiated from iPSC colonies through the mesoderm and hematopoietic stem cell stages to NK cells (day 26 of differentiation). [Figure 18a] 18a and 18b show the results of flow cytometry analysis of cells differentiated from iPSC colonies through the mesodermal and hematopoietic stem cell stages to NK cells (day 47 of differentiation). [Figure 18b] 18a and 18b show the results of flow cytometry analysis of cells differentiated from iPSC colonies through the mesodermal and hematopoietic stem cell stages to NK cells (day 47 of differentiation). [Figure 19] FIG. 19 shows a method for differentiation of iPSCs into natural killer cells using Stempro34 medium. [Figure 20] FIG. 20 shows the results of flow cytometry analysis of cells at the mesodermal cell stage (day 4 of differentiation) during differentiation of iPSC colonies into NK cells according to the method of FIG. [Figure 21] FIG. 21 shows the results of flow cytometry analysis of cells differentiated through the mesodermal cell stage to hematopoietic stem cells (day 12 of differentiation) during differentiation of iPSC colonies to NK cells according to the method of FIG. [Figure 22] FIG. 22 shows the results of flow cytometry analysis of cells differentiated from iPSC colonies through the mesoderm and hematopoietic stem cell stages to NK cells (day 26 of differentiation) according to the method of FIG. [Figure 23] FIG. 23 shows the results of flow cytometry analysis of cells differentiated from iPSC colonies through the mesoderm and hematopoietic stem cell stages to NK cells (day 30 of differentiation) according to the method of FIG. [Figure 24] FIG. 24 shows the results of flow cytometry analysis of cells differentiated from iPSC colonies through the mesoderm and hematopoietic stem cell stages to NK cells (day 34 of differentiation) according to the method of FIG. [Diagram 25] FIG. 25 shows the results of flow cytometry analysis of cells differentiated from iPSC colonies through the mesoderm and hematopoietic stem cell stages to NK cells (day 42 of differentiation) according to the method of FIG. [Figure 26] FIG. 26 is a graph showing the results obtained by confirming the cytotoxicity of iPSC-derived NK cells (EiNK cells) through co-culture with K562 cells, a chronic myeloid leukemia cell line. [Figure 27] FIG. 27 is a schematic diagram of a feeder cell-based expansion method for iPSC-derived NK cells (EiNK cells). [Figure 28] FIG. 28 shows the results obtained by examining morphological changes at various time points during the mass expansion process of iPSC-derived NK cells (EiNK cells) using an optical microscope. [Figure 29] FIG. 29 is a graph showing the results obtained by measuring the cell number at each time point during the mass expansion process of iPSC-derived natural killer cells (EiNK cells) as expansion rate and cell number. [Figure 30a] 30a and 30b are a diagram and a graph showing the results obtained by confirming NK cell activity by flow cytometry after massive expansion of iPSC-derived NK cells (EiNK cells) for 20 days. [Figure 30b] 30a and 30b are a diagram and a graph showing the results obtained by confirming NK cell activity by flow cytometry after massive expansion of iPSC-derived NK cells (EiNK cells) for 20 days. [Diagram 31] FIG. 31 shows graphs and tables comparing the results obtained by determining the cytotoxicity of NK cells against tumor cells after bulk expansion of iPSC-derived NK cells (EiNK cells) for 21 or 28 days. [Diagram 32]FIG. 32 shows the results obtained by confirming the expression of induced pluripotent stem cell marker proteins (TRA-1-60, SSEA4) by flow cytometry after massive expansion of iPSC-derived NK cells (EiNK cells) for 21 days. [Diagram 33] FIG. 33 is a graph showing the results obtained by determining the cell proliferation rate during long-term culture of iPSC-derived NK cells (EiNK cells). [Figure 34a] Figures 34a and 34b are diagrams and graphs showing the results obtained by determining the expression of marker proteins of NK cells (CD3, CD45) or induced pluripotent stem cells (TRA-1-60, SSEA4) (Figure 35a, 63 days of culture); and the tumor cell killing ability of iPSC-derived NK cells (EiNK cells) after culturing for different periods (20 days, 56 days, and 63 days) (Figure 35b). [Figure 34b] Figures 34a and 34b are diagrams and graphs showing the results obtained by determining the expression of marker proteins of NK cells (CD3, CD45) or induced pluripotent stem cells (TRA-1-60, SSEA4) (Figure 35a, 63 days of culture); and the tumor cell killing ability of iPSC-derived NK cells (EiNK cells) after culturing for different periods (20 days, 56 days, and 63 days) (Figure 35b). [Figure 35a] Figures 35a and 35b are a diagram and a graph showing the results obtained by confirming the expression of cell surface proteins in iPSC-derived NK cells (EiNK cells, cultured for 21 days) and blood-derived NK cells (PBNK cells, cultured for 22 days) by flow cytometry. [Figure 35b] Figures 35a and 35b are a diagram and a graph showing the results obtained by confirming the expression of cell surface proteins in iPSC-derived NK cells (EiNK cells, cultured for 21 days) and blood-derived NK cells (PBNK cells, cultured for 22 days) by flow cytometry. [Diagram 36]FIG. 36 is a graph showing the results obtained by examining the expression of genes associated with tumor killing and activation of NK cells in iPSC-derived NK cells (EiNK cells, cultured for 28 days) and blood-derived NK cells (PBNK cells, cultured for 22 days). [Figure 37a] Figures 37a and 37b are graphs showing cytokines selected to have 2-fold or higher (37a) or 1.3-1.9-fold or higher (37b) concentrations in the culture medium of EiNK cells by measuring cytokines in the culture medium of NK cells from iPSC-derived NK cells (EiNK cells, cultured for 21 days) and blood-derived NK cells (PBNK cells, cultured for 21 days). [Figure 37b] Figures 37a and 37b are graphs showing cytokines selected to have 2-fold or higher (37a) or 1.3-1.9-fold or higher (37b) concentrations in the culture medium of EiNK cells by measuring cytokines in the culture medium of NK cells from iPSC-derived NK cells (EiNK cells, cultured for 21 days) and blood-derived NK cells (PBNK cells, cultured for 21 days). [Figure 38] Figure 38 shows the results obtained by confirming the expression of natural killer cell inhibitory receptors in iPSC-derived NK cells (EiNK cells, cultured for 21 days) and blood-derived NK cells (PBNK cells, cultured for 21 days) by flow cytometry. [Figure 39] Figure 39 is a graph showing the results obtained by confirming the tumor cell-killing ability of iPSC-derived NK cells (EiNK cells, cultured for 21 or 28 days) and blood-derived NK cells (PBNK cells, cultured for 16 days) by flow cytometry. [Figure 40a] Figures 40a-40e show the results obtained by checking the luciferase activity in NSG mice at different time periods after administration of a luciferase-expressing ovarian cancer (OVCAR3) cell line followed by treatment with iPSC-derived NK cells (EiNK cells, cultured for 21 days) (40a-40d) and the graph showing the results obtained by quantifying said activity (40e). [Figure 40b] Figures 40a-40e show the results obtained by checking the luciferase activity in NSG mice at different time periods after administration of a luciferase-expressing ovarian cancer (OVCAR3) cell line followed by treatment with iPSC-derived NK cells (EiNK cells, cultured for 21 days) (40a-40d) and the graph showing the results obtained by quantifying said activity (40e). [Figure 40c] Figures 40a-40e show the results obtained by checking the luciferase activity in NSG mice at different time periods after administration of a luciferase-expressing ovarian cancer (OVCAR3) cell line followed by treatment with iPSC-derived NK cells (EiNK cells, cultured for 21 days) (40a-40d) and the graph showing the results obtained by quantifying said activity (40e). [Figure 40d] Figures 40a-40e show the results obtained by checking the luciferase activity in NSG mice at different time periods after administration of a luciferase-expressing ovarian cancer (OVCAR3) cell line followed by treatment with iPSC-derived NK cells (EiNK cells, cultured for 21 days) (40a-40d) and the graph showing the results obtained by quantifying said activity (40e). [Figure 40e] Figures 40a-40e show the results obtained by checking the luciferase activity in NSG mice at different time periods after administration of a luciferase-expressing ovarian cancer (OVCAR3) cell line followed by treatment with iPSC-derived NK cells (EiNK cells, cultured for 21 days) (40a-40d) and the graph showing the results obtained by quantifying said activity (40e). [Diagram 41] FIG. 41 is a graph showing results obtained by administering a luciferase-expressing ovarian cancer (OVCAR3) cell line to NSG mice and then determining mouse survival after treatment with iPSC-derived NK cells (EiNK cells, cultured for 21 days). [Figure 42a]Figures 42a to 42c show the results obtained by checking the yield of cells obtained after CD34 sorting (Figure 42a), cell death (Figure 42a), expression of NK cell markers ( / activation markers) (Figure 42b), and cell proliferation rate (Figure 42c), and Figure 42d shows the results obtained by checking the expression of hematopoietic stem cell marker proteins (CD34 / CD45) by flow cytometry without an isolation step with the CD34 marker during EiNK cell differentiation, as one embodiment of the EiNK cell differentiation method of the present invention. [Figure 42b] Figures 42a to 42c show the results obtained by checking the yield of cells obtained after CD34 sorting (Figure 42a), cell death (Figure 42a), expression of NK cell markers ( / activation markers) (Figure 42b), and cell proliferation rate (Figure 42c), and Figure 42d shows the results obtained by checking the expression of hematopoietic stem cell marker proteins (CD34 / CD45) by flow cytometry without an isolation step with the CD34 marker during EiNK cell differentiation, as one embodiment of the EiNK cell differentiation method of the present invention. [Fig. 42c] Figures 42a to 42c show the results obtained by checking the yield of cells obtained after CD34 sorting (Figure 42a), cell death (Figure 42a), expression of NK cell markers ( / activation markers) (Figure 42b), and cell proliferation rate (Figure 42c), and Figure 42d shows the results obtained by checking the expression of hematopoietic stem cell marker proteins (CD34 / CD45) by flow cytometry without an isolation step with the CD34 marker during EiNK cell differentiation, as one embodiment of the EiNK cell differentiation method of the present invention. [Fig.42d]Figures 42a to 42c show the results obtained by checking the yield of cells obtained after CD34 sorting (Figure 42a), cell death (Figure 42a), expression of NK cell markers ( / activation markers) (Figure 42b), and cell proliferation rate (Figure 42c), and Figure 42d shows the results obtained by checking the expression of hematopoietic stem cell marker proteins (CD34 / CD45) by flow cytometry without an isolation step with the CD34 marker during EiNK cell differentiation, as one embodiment of the EiNK cell differentiation method of the present invention. [Diagram 43] FIG. 43 is a graph showing the results obtained by measuring the size of embryoid bodies (EBs) formed at each culture period during the third culture of iPSCs using a conventional EiNK cell generation method. [Diagram 44] FIG. 44 is a graph showing the results obtained by measuring the viability of batch-isolated cells after isolating cells from embryoid bodies (EBs) formed through the third culture of iPSCs by a conventional EiNK cell generation method. [Diagram 45] FIG. 45 is a graph showing the results obtained by comparing the time period required for EiNK cell production according to a conventional EiNK cell production method and the EiNK cell production method according to one embodiment of the present invention. [Diagram 46] Figure 46 shows the results obtained by confirming by flow cytometry the expression of marker proteins of natural killer cells (CD56, CD45, NKp30, NKp44) and induced pluripotent stem cells (TRA-1-60, SSEA4) in EiNK cells produced according to the EiNK cell production method of one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Natural killer cells In one aspect of the present invention, there is provided a natural killer cell expressing any one protein selected from the group consisting of CD25, CD9, CD80, and combinations thereof. The natural killer cell may further express any one protein selected from the group consisting of CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, CD210, and combinations thereof.

[0012] As used herein, the term "natural killer cells (NK cells)" refers to cytotoxic lymphocytes that constitute a major component of the innate immune system, defined as large granular lymphocytes (LGLs) and constituting the third cell type distinct from B and T lymphocytes that are generated from common lymphoid progenitors (CLPs). Lymphocytes play a role in immune responses, are present in about 10-15% of normal human blood, and have high killing ability when reacting with non-self. Natural killer cells may respond non-specifically and immediately to various virus-infected cells, bacterial invasion, or the occurrence of abnormal cells to remove foreign substances. Natural killer cells are activated in response to interferon or macrophage-derived cytokines, and natural killer cells contain two types of extracellular receptors that control the cytotoxic activity of the cells, represented by "activating receptors" and "inhibitory receptors".

[0013] As used herein, the term "CD25" refers to the alpha subunit of the interleukin-2 receptor, which is known to regulate the activity of regulatory T cells (Tregs) and suppress the activity and proliferation of activated T cells, thereby regulating the immune response.

[0014] As used herein, the term "CD80 (cluster of differentiation 80)" is also known as B7-1. CD80 is a cell surface protein belonging to the B7 family of the immunoglobulin superfamily. CD80 consists of an Ig-like extracellular domain (one variable and one constant), a transmembrane region, and a cytoplasmic domain. The costimulatory ligand CD80 is expressed on antigen-presenting cells (APCs) such as monocytes, dendritic cells, and activated B cells, as well as on T cells. CD80 binds to CD28 or CTLA-4 (cytotoxic T lymphocyte antigen-4) to regulate cell proliferation and IL-2 production. When CD80 binds to CD28, it activates T cells to promote immune responses, whereas when CD80 binds to the immune checkpoint protein CTLA-4, it reduces the activity of T cells and suppresses immune responses.

[0015] As used herein, the term "CD9" refers to a protein belonging to the transmembrane protein 4 superfamily, also known as the tetraspanin family, which is involved in integrin-associated sperm-egg fusion, platelet activation and aggregation, cell adhesion, and cell migration.

[0016] As used herein, the term "CD1d" is an antigen-presenting protein that binds to self and non-self glycolipids and presents the glycolipids to the T cell receptor of natural killer T cells (NKT cells).

[0017] As used herein, the term "CD30" is also known as TNFRSF8, a protein expressed in activated T cells and B cells. CD30 is known to induce apoptosis by binding to TRAF2 or TRAF5 and activating the NF-κB signaling pathway. In addition, CD30 suppresses autoimmunity by suppressing the proliferation and activity of autoactivated cytotoxic T cells.

[0018] As used herein, the term "CD87" is also known as the urokinase receptor or uPAR (urokinase plasminogen activator surface receptor) and plays a role in promoting plasmin formation and localization. Plasmin is a serine protease that acts to dissolve fibrin clots, and cell surface-bound plasmin is known to promote ECM degradation by activating MMPs that can degrade collagen and other proteins in the blood vessel wall and is involved in the migration of neutrophils, monocytes, and macrophages.

[0019] As used herein, the term "CDw327" is known as SIGLEC6 (sialic acid-binding Ig-like lectin 6) or CD33LSIGLEC6, a cell adhesion protein involved in sialic acid-dependent cell binding.

[0020] As used herein, the term "CD33," also known as Siglec-3, is a receptor expressed on cells of the myeloid lineage. CD33 binds to any molecule that contains sialic acid residues, such as glycoproteins or glycolipids, and upon activation induces a signaling pathway that inhibits phagocytosis.

[0021] As used herein, the term "CD49a" refers to integrin alpha-1 (integrin α-1), a subunit of the integrin α1β1 receptor.

[0022] As used herein, the term "CD49c" refers to integrin alpha-3, a subunit of the integrin α3β1 receptor that is involved in neuronal migration and corticogenesis.

[0023] As used herein, the term "CD49e" refers to integrin alpha-5, a subunit of the integrin α5β1 receptor. α5 is post-translationally cleaved in the extracellular domain and binds to β1 to form the fibronectin receptor.

[0024] As used herein, the term "CD49f" refers to integrin alpha-6, which binds to integrin beta 4 (β4), also known as TSP180, or to β1 of integrin VLA-6, thereby mediating cell adhesion and cell signaling.

[0025] As used herein, the term "CD66a" is also known as CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) and belongs to the CEA (carcinoembryonic antigen) superfamily. This protein acts as both an inhibitory and an activating receptor. For example, CD66a acts as an inhibitory receptor in immune response and insulin action, and has the function of promoting angiogenesis. CEACAM1 is known to suppress immune responses by suppressing T cell proliferation and cytokine production, suppressing natural killer cell activity, and suppressing IL-1β secretion from neutrophils.

[0026] As used herein, the term "CD66c" is also known as CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6) and belongs to the CEA (carcinoembryonic antigen) superfamily. CD66c is known to be involved in cell adhesion and tumor formation / progression.

[0027] As used herein, the term "CD66d" is also known as CEACAM3 (carcinoembryonic antigen-related cell adhesion molecule 3) and belongs to the CEA (carcinoembryonic antigen) superfamily. CD66d is expressed on granulocytes and is known to regulate phagocytosis against pathogens in natural immunity.

[0028] As used herein, the term "CD66e" is also known as CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5) and belongs to the CEA (carcinoembryonic antigen) superfamily. CD66e regulates cell adhesion, cell signaling, and tumorigenesis / progression.

[0029] As used herein, the term "CD71" is also known as TFR1 (transferrin receptor protein 1), a protein involved in the influx of ions into cells via transferrin. CD71 is known to be expressed only on normal hematopoietic bone marrow and splenic erythroid precursors.

[0030] As used herein, the term "CD83" is used as a marker protein for mature dendritic cells. CD83 is known to stabilize the expression of MHCII, costimulatory molecules, and CD28 on the dendritic cell membrane by suppressing the activity of E3 ubiquitin ligase.

[0031] As used herein, the term "CDw93" is also known as C1q binding protein (C1qRp). CDw93 is expressed in monocytes (macrophages) and neutrophils, but not in T cells and B cells. CDw93 binds to C1q, the recognition subunit of the first component (C1) of the complement pathway, and promotes C1q-mediated phagocytosis. CDw93 is known to be highly expressed in stem cells (progenitors) that differentiate into hematopoietic cells and hepatocytes.

[0032] As used herein, the term "CD106" is also referred to as VCAM-1 (vascular cell adhesion molecule 1), which mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. Additionally, CD106 mediates the interaction between leukocytes and vascular endothelial cells through signal transduction. CD106 serves as an endothelial ligand for VLA-4 (very late antigen-4 or integrin α4β1).

[0033] As used herein, the term "CD108" refers to SEMA7A (Semaphorin 7A), also known as JMH (John-Milton-Hagen) blood group antigen. CD108 is expressed on activated lymphocytes and erythrocytes, and is also found in the esophagus, heart, brain, spleen, testis, lung, ovary, and uterus. CD108 plays an important role in integrin-mediated signaling, regulating cell migration and immune response.

[0034] As used herein, the term "CD114" is known as GCSFR (granulocyte colony-stimulating factor receptor), which is the cell surface receptor for GCSF. CD114 is a type 1 cytokine receptor that belongs to the hematopoietin receptor superfamily and is expressed on progenitor cells present in bone marrow, where it mediates cell proliferation and differentiation of progenitor cells into mature neutrophils and macrophages.

[0035] As used herein, the term "CD123" is the interleukin-3 receptor, which is expressed on basophils and dendritic cells and mediates the IL-3 mediated immune response.

[0036] As used herein, the term "CD124" refers to the interleukin-4 receptor and belongs to the type 1 cytokine receptors. CD124 binds IL-4 and IL-13 and regulates the immune response by mediating IgE antibody production, promoting Th2 cell differentiation, and the like.

[0037] As used herein, the term "CD146" is known as MCAM (melanoma cell adhesion molecule) and is used as a marker protein of vascular endothelial cell lineage. CD146 is a receptor for laminin alpha 4 (laminin α4) and is abundantly expressed in cells that compose the vascular wall, such as vascular endothelial cells, smooth muscle cells, and pericytes. CD146 is also known to be expressed in some subgroups of T cells and B cells.

[0038] As used herein, the term "CD150" is also known as SLAMF1 (signaling lymphocyte activation molecule 1), which mediates interactions between homotypic and heterotypic cells, thereby regulating various immune cell activities and differentiation.

[0039] As used herein, the term "CD164" is also known as endrin or sialomucin core protein 24. Sialomucin is a protein involved in both cell adhesion inhibition and cell adhesion, and CD164 is a protein involved in cell adhesion.

[0040] As used herein, the term "CD196" is also known as chemokine receptor 6 (CCR6) and belongs to the G protein-coupled receptor superfamily. CD196 is expressed on B cells, T cells (Th1, Th2, Th17, Treg), natural killer T cells (NKT cells), immature dendritic cells, and neutrophils. CD196 binds to CCL20 (MIP3α) and regulates immune responses by mediating B cell maturation and differentiation, dendritic cell and T cell migration, and the like.

[0041] As used herein, the term "CD210" refers to a receptor for interleukin-10 and belongs to the type 2 cytokine receptor. CD210 is composed of α and β subunits, and the α subunit is expressed in hematopoietic cells such as T cells, B cells, natural killer cells, mast cells, and dendritic cells. Meanwhile, the expression of the β subunit is largely regulated by ubiquitin. The α subunit is the ligand-binding site of the IL-10 receptor and is present only in the IL-10 receptor, while the β subunit also binds to the subunits of other type 2 cytokine receptors to form receptors. The IL-10 receptor is known to mediate gastrointestinal immune responses mainly in the mucosal layer.

[0042] In the present invention, expression of the above CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196 and CD210 proteins may be increased compared to natural killer cells isolated from blood. Specifically, expression of the above CD25, CD9, CD80, CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, and CD210 proteins may be increased by about at least two-fold compared to natural killer cells isolated from blood.

[0043] In this case, the natural killer cells isolated from blood may be from mammals such as rats, mice, livestock, and humans. The natural killer cells isolated from blood may be natural killer cells isolated from blood without further modification or natural killer cells that have been induced to be activated. Preferably, the natural killer cells isolated from blood may be natural killer cells that have been induced to be activated.

[0044] As used herein, the term "activated natural killer cells" may refer to natural killer cells that have increased cytotoxicity or enhanced intrinsic immunomodulatory capabilities compared to natural killer cells isolated from blood without further modification. Activated natural killer cells may overexpress receptors that can target the cells or tissues to be treated.

[0045] The method for activating natural killer cells may be to culture natural killer cells isolated from blood under certain conditions, genetically modify natural killer cells, or treat natural killer cells with a substance that increases the activity of natural killer cells. In this case, natural killer cell culture may use conventional animal cell culture media known in the art, such as, but not limited to, CellGro medium (Cellgenix), AIM-V medium, XVIVO 20, RPMI1640, and the like.

[0046] As used herein, the term "substance that increases the activity of natural killer cells" refers to any substance that can increase the activity of natural killer cells, for example, the cytotoxicity of natural killer cells, or generate, increase, or proliferate activated natural killer cells. Substances that increase the activity of natural killer cells may include, but are not limited to, immunocytokines, so long as they are substances that can increase the activity of natural killer cells.

[0047] As used herein, the term "cytokine" refers to a signaling substance that controls the body's defense system and stimulates the body, and may refer to a physiologically active regulatory protein. Cytokines regulate various biological activities, such as cell activation, differentiation, cell migration, senescence, and apoptosis induction in various cells through autocrine and paracrine actions.

[0048] The cytokine may be from the interferon (IFN) family, the interleukin family, the tumor necrosis factor (TNF) family, or a combination thereof.

[0049] The interferon may be any one selected from the group consisting of type 1 interferon (e.g., interferon-α, interferon-β, interferon-κ, interferon-ω), type 2 interferon (e.g., interferon-γ), type 3 interferon, and combinations thereof. The interleukin may be any one selected from the group consisting of IL-2, IL-5, IL-8, IL-12, IL-15, IL-18, IL-21, and combinations thereof. In this case, the TNF may include TNF-α, TNF-β, or TNF-γ, etc. The immunocytokine may include a cytokine-antibody complex. For example, the immunocytokine may include an interleukin-anti-interleukin antibody complex.

[0050] Specifically, the natural killer cells of the present invention are obtained by culturing blood-derived natural killer cells for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or 22 days.

[0051] The activated natural killer cells of the present invention may express any one natural killer cell marker protein selected from the group consisting of CD56, CD45, CD16, NKp30, NKp44, NKG2D, DNAM1, and combinations thereof in addition to the above proteins, and may not express CD3, a marker protein of T cells, or may express CD3 at a lower level compared to T cells. Here, natural killer cell activation is the same as above, and whether the natural killer cells are activated may be evaluated by detecting one or more functionally related markers, such as CD56, CD45, CD16, NKp30, NKp44, NKG2D, DNAM1, and the like.

[0052] Furthermore, the natural killer cells according to the present invention may have increased expression of genes related to the tumor-killing ability of natural killer cells and natural killer cell activation compared to natural killer cells isolated from blood. Specifically, the natural killer cells according to the present invention may be characterized by further increased expression of any one gene selected from the group consisting of GZMB, CD226, RASGRP1, CD107a, CCR9, CD40LG, CD300c, and combinations thereof compared to natural killer cells isolated from blood. In this case, the natural killer cells obtained from blood are the same as above.

[0053] As used herein, the term "GZMB (granzyme B)" refers to one of the serine proteases expressed in cytotoxic T cells and natural killer cells. T cells and natural killer cells induce apoptosis of target cells through granzyme B.

[0054] As used herein, the term "CD226" is an immunoglobulin-like transmembrane glycoprotein known as PTA1 (platelet and T-cell activation antigen 1) or DNAM-1 (DNAX accessory molecule-1). CD226 is expressed on natural killer cells, natural killer T cells, B cells, dendritic cells, hematopoietic stem cells, platelets, monocytes, and T cells. CD226 binds to CD112 or CD155 and is involved in cell adhesion. CD226 mediates lymphocyte signaling and regulates lymphokine secretion and cytotoxicity of natural killer cells and cytotoxic T cells. In addition, CD226 regulates platelet activation and aggregation, and transendothelial migration of natural killer cells.

[0055] As used herein, the term "RASGRP1 (RAS guanyl releasing protein 1)" is a protein belonging to the Ras superfamily that contains a guanine nucleotide exchange factor (GEF) domain and regulates the development, homeostasis, and differentiation of T and B cells.

[0056] As used herein, the term "CD107a" is also known as LAMP-1 (lysosome-associated membrane protein 1 or lysosome-associated membrane glycoprotein 1), LGP120, and LAMPA, and is a degranulation marker of cytotoxic T cells and natural killer cells. CD107a mediates the translocation of cytotoxic granules to the cell surface and the adhesion of perforin to cytotoxic granules, thereby regulating the cytolytic activity of target cells. In addition, CD107a acts to protect natural killer cells from damage caused by the degranulation process.

[0057] As used herein, the term "CCR9" refers to the receptor for CCL25, also known as CDw199. CCR9 is known to be involved in cancer growth and inflammatory diseases by binding to CCL25. CCR9 / CCL25 interaction promotes tumor cell growth and metastasis and inhibits cell death, thereby promoting cancer progression. Additionally, CCR9 is known to promote the development of inflammatory diseases (inflammatory bowel disease, cardiovascular disease, rheumatoid arthritis, asthma, and the like).

[0058] As used herein, the term "CD40LG (or CD40L)" is also known as CD154 and is a protein expressed on T cells. CD40LG is a costimulatory molecule that binds to CD40 expressed on antigen-presenting cells and activates immune cells.

[0059] As used herein, the term "CD300c" is also known as CMRF-35 or CMRF-35A. The B7 family is a member of the Ig superfamily and is known to be expressed on the surface of antigen-presenting cells such as B cells, macrophages, and dendritic cells. Suppression of the expression or activity of CD300c protein induces T cell activation and / or promotes differentiation of M1 macrophages.

[0060] The natural killer cells according to the invention may be characterized by increased secretion of any one of the proteins selected from the group consisting of FLT3L, MIP1 alpha / beta, PF4, IGFBP2, IGFBP3, MMP9, and combinations thereof, compared to natural killer cells isolated from blood, in which case the protein is a secretory protein secreted to the outside of the cell, and the secretion may be increased at least two-fold in the natural killer cells according to the invention, compared to natural killer cells isolated from blood, in which case the natural killer cells obtained from blood are the same as above.

[0061] As used herein, the term "FLT3L (FMS-like tyrosine kinase 3 ligand)" refers to a protein encoded by the FLT3LG gene in humans. FLT3L is structurally similar to stem cell factor (SCF) and colony-stimulating factor 1 (CSF-1), and promotes the proliferation and differentiation of hematopoietic stem cells together with various regulatory factors.

[0062] As used herein, the term "MIP1 (macrophage inflammatory protein 1)" is a cc motif chemokine, which exists in two forms in humans: MIP-1α and MIP-1β. MIP-1α and MIP-1β, also known as CCL3 and CCL4, respectively, are secreted by activated macrophages and monocytes. CCL3 functions as a ligand for CCR1, and CCL4 functions as a ligand for CCR1 and CCR5, thereby activating monocytes, T cells, dendritic cells, NK cells, and platelets, etc.

[0063] As used herein, the term "PF4 (platelet factor 4)" is also known as CXC4 (chemokine CXC ligand 4), and is a ligand for CXCR3-B. PF4 is released from the alpha granules of activated platelets during platelet aggregation and promotes blood clotting.

[0064] As used herein, the term "IGFBP2 (insulin-like growth factor binding protein 2)" refers to a protein that binds to IGF protein with high affinity. IGFBP2 inhibits the binding of IGF to IGF receptor (IGFR) through the binding of IGFBP2 to IGF, thereby inhibiting cell proliferation and growth through IGF / IGFR interaction.

[0065] As used herein, the term "IGFBP3" refers to a protein that binds with high affinity to IGF-1 or IGF-2. IGFBP3 is known to bind to IGF in the blood and play a role in extending its transport and half-life.

[0066] As used herein, the term "MMP9 (matrix metallopeptidase 9)" is also known as type 4 collagenase having 92 kDa or gelatinase B (GELB). MMP9 is induced to be expressed by inflammatory stimuli and promotes cell migration by degrading the matrix.

[0067] The natural killer cells of the present invention may be natural killer cells that do not express any one protein selected from the group consisting of CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM3, TIGIT, and combinations thereof, or that express these proteins at a lower level than natural killer cells obtained from blood. Specifically, the natural killer cells may be natural killer cells that express any one protein selected from the group consisting of CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM3, TIGIT, and combinations thereof at a level half or less than that of natural killer cells obtained from blood. In this case, the natural killer cells obtained from blood are the same as those described above.

[0068] As used herein, the term "CD205," also known as lymphocyte antigen 75 or DEC-205, is a receptor involved in the endocytosis of antigens captured on the outside of cells. CD205 is known to reduce B cell proliferation.

[0069] As used herein, the term "CD158b" is known as KIR2DL2 / DL3 (Killer Cell Immunoglobulin-Like Receptor 2DL2 / DL3) or two Ig domains and long cytoplasmic tail 2, and is a receptor for HLA-C (HLA-Cw1, HLA-Cw3, HLA-Cw7) present on natural killer cells. This protein is known to inhibit the activity of natural killer cells and inhibit the destruction of cancer cells by natural killer cells.

[0070] As used herein, the term "CD243 (cluster of differentiation 243)" is also known as P-gp (permeability glycoprotein, P-glycoprotein 1, Pgp), MDR1 (multidrug resistance protein 1), or ABCB1 (ATP-binding cassette subfamily B member 1), and is a cell membrane protein that expels foreign substances (toxins or drugs) from the inside of the cell to the outside of the cell in an ATP-dependent manner. CD243 is widely distributed in the intestinal epithelium and is highly expressed in some cancer cells, and is also known to cause anticancer drug resistance.

[0071] As used herein, the term "γδ TCR" is a T cell antigen receptor (TCR) expressed on γδ T cells. While the antigen receptor of αβ T cells (αβ TCR) recognizes peptide antigens presented by major histocompatibility complex molecules (pMHC) on antigen presenting cells (APCs), γδ TCR is known to partially recognize stress-induced self-antigens, lipids, or pyrophosphates secreted in a somewhat restricted manner by some microorganisms or overproduced by tumor cells.

[0072] As used herein, the term "NKB1" is also known as KIR3DL1 (killer cell immunoglobulin-like receptor 3DL1), NKAT3, or MHC class I NK cell receptor. NKB1 is expressed on natural killer cells and acts as a receptor for HLA-Bw4. NKB1 is known to suppress the activity of natural killer cells and to suppress the lysis of tumor cells by natural killer cells.

[0073] As used herein, the term "CD158a" is also known as KIR2DS1 (Killer Cell Immunoglobulin-Like Receptor 2DS1), and is a receptor for HLA-C (HLA-Cw6) present in natural killer cells. Unlike CD158b, which inhibits the activity of natural killer cells, this protein does not inhibit the activity of natural killer cells.

[0074] As used herein, the term "CDw328" is also known as SIGLEC7 (sialic acid-binding Ig-like lectin 7), a cell adhesion protein involved in sialic acid-dependent cell binding.

[0075] As used herein, the term "TIM-3 (T cell immunoglobulin and mucin domain containing-3), also known as HAVCR2 (Hepatitis A virus receptor 2), is an immune checkpoint protein that suppresses immune responses. TIM-3 is expressed on a variety of tumor cells, including T cells, monocytes, macrophages, dendritic cells, mast cells, and natural killer cells. The ligands for TIM-3 are known to be galectin-9, PtdSer (phosphatidylserine), HMGB1 (high mobility group box 1), and CEACAM1.

[0076] As used herein, the term "TIGIT (T cell immunoreceptor with Ig and ITIM domains)" is also known as WUCAM and Vstm2, and is an immunoreceptor present on some T cells and natural killer cells. TIGIT binds to CD155 present on dendritic cells, macrophages, and the like, and suppresses the activity of T cells and natural killer cells throughout the body, thereby reducing the immune response.

[0077] In the present invention, natural killer cells can be induced to differentiate from induced pluripotent stem cells. Specifically, a method for producing natural killer cells includes culturing induced pluripotent stem cells (iPSCs) in a cell culture vessel to a depth of 1 cm. 2 The natural killer cells of the present invention can be produced by a method including the steps of seeding up to 0.5 to 500 cells per area of ​​a cell culture vessel and performing adherent culture to produce iPSC colonies; differentiating the iPSC colonies into mesodermal cells to produce mesodermal cells; differentiating the mesodermal cells into hematopoietic stem cells to produce hematopoietic stem cells; and differentiating the hematopoietic stem cells into natural killer cells to produce natural killer cells.

[0078] Furthermore, in the present invention, natural killer cells can be produced by further carrying out mass culture expansion of the induced pluripotent stem cells.

[0079] Specifically, the natural killer cells of the present invention can be induced to differentiate from induced pluripotent stem cells through the following steps.

[0080] First, the method is 2 The method includes a step of seeding induced pluripotent stem cells (iPSCs) as iPSCs or iPSC spheroids into a cell culture vessel so that 0.5 to 20 colonies are formed per area of ​​the cell culture vessel, and performing adherent culture to produce iPSC colonies.

[0081] As used herein, the term "stem cell" refers to a relatively undifferentiated cell that has not yet developed and has the ability to differentiate into a specific tissue cell under appropriate conditions. A stem cell can self-renew and generate daughter cells that have the ability to maintain their own undifferentiated characteristics and at the same time generate daughter cells that differentiate into a specific cell type. Stem cell marker proteins include Lgr5, Lrp4, Msi-1, Sca-1, SCF, SSEA-4, Tra-1-60, Tra-1-81, and the like. Stem cells include embryonic stem cells, adult stem cells, and induced pluripotent stem cells.

[0082] As used herein, the term "induced pluripotent stem cells (iPSCs)" refers to cells generated by inducing non-pluripotent somatic cells to dedifferentiate into pluripotent cells, such as embryonic stem cells. Induced pluripotent stem cells may also be referred to as dedifferentiated stem cells. Induced pluripotent stem cells may be generated using somatic cells from fetuses, neonates, children, or adults. Induced pluripotent stem cells may be derived from fibroblasts, seratinocytes, blood cells, or kidney epithelial cells. Induced pluripotent stem cells may be generated by reprogramming somatic cells. Methods for reprogramming somatic cells to generate induced pluripotent stem cells may be performed using known methods, such as those described in Takahashi K, Yamanaka S (2006), Cell. 126:663-676.

[0083] As used herein, the term "differentiation" refers to the phenomenon of cell division, proliferation, and specialization of cellular structure or function during the development of an entire organism. In other words, differentiation is the process by which cells, tissues, and the like of living organisms change to have the morphology and function appropriate for their respective roles. In the present invention, the term includes the process by which induced pluripotent stem cells transform into natural killer cells, and may include the process by which progenitor cells express specific differentiation traits.

[0084] The number of cells or spheroids to be seeded on a 1 cm 2 The range is 0.5 to 20 per square centimeter.2 The number of iPSCs or iPSC spheroids per area may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 iPSCs or iPSC spheroids per area.

[0085] In this case, the iPSC spheroid may contain about 30 to about 600 iPSCs. The iPSC spheroid may further contain about 50 to about 300 iPSCs. The iPSC spheroid may further contain about 70 to about 200 iPSCs. The iPSC spheroid may further contain about 80, about 90, about 100, or about 120 iPSCs.

[0086] Additionally, the number of cells seeded may be calculated in different ways. The number of cells present per ml of culture medium can be calculated per cm by using the method for calculating the number of cells seeded. 2 The amount of cells per well may be determined as the number of cells per well.

[0087] Specifically, the number of induced pluripotent stem cells seeded may be about 0.5 to about 2,500 per mL of culture medium. Furthermore, the number of cells may be about 1 to about 2,500, about 10 to about 2,500, about 20 to about 2,500, about 30 to about 2,500, about 40 to about 2,500, about 50 to about 2,500, about 60 to about 2,500, about 70 to about 2,500, about 80 to about 2,500, about 90 to about 2,500, about 100 to about 2,500, about 200 to about 2,500, about 300 to about 2,500, about 400 to about 2,500, about 500 to about 2,500, about 600 to about 2,500, about 700 to about 2,500, about 800 to about 2,500, about 900 to about 2,500, or about 1,000 per mL. Preferably, the number of cells may be about 250 to about 2,500.

[0088] The total number of induced pluripotent stem cells seeded by the above method on the day of differentiation initiation was 1 cm 2 Approximately 0.5 to 1 × 10 per cell culture vessel area 7 Specifically, the total number of induced pluripotent stem cells seeded by the above method on the day of initiation of differentiation is 1 cm 2 Approximately 1 to 5 × 10 per cell culture vessel area6 , about 10 to about 2 x 10 6 , about 1×10 2 ~Approx. 1×10 6 , about 1×10 3 ~Approx. 5×10 5 , or about 1 × 10 4 ~Approx. 1×10 5 Preferably, the total number of induced pluripotent stem cells seeded by the above method on the day of initiation of differentiation is 1 cm 2 Approximately 1 x 10 per cell culture vessel area 3 ~Approx. 1×10 6 That's fine too.

[0089] As used herein, the term "culture" means growing cells under appropriately controlled environmental conditions, and the culture step of the present invention may be carried out in an appropriate medium known in the art and under appropriate culture conditions. This culture step may be easily adjusted and used by those skilled in the art depending on the selected cells. The above-mentioned "medium" means a known medium used to culture cells, and includes all known cell culture media or modified media thereof.

[0090] iPSCs may be cultured in a medium containing a ROCK inhibitor. The ROCK inhibitor may be selected from the group consisting of Y-27632, thiazovivin, fasudil, GSK429286A, RKI-1447, H-1152, and azaindole 1. iPSCs may be cultured in a medium containing a ROCK inhibitor for about 10 minutes to about 1 week, about 30 minutes to about 6 days, about 1 hour to about 5 days, about 2 hours to about 4 days, about 3 hours to about 3 days, about 6 hours to about 2 days, about 12 hours to about 2 days, or about 18 hours to about 1 day.

[0091] iPSCs may be cultured by adherent culture. After the single cell iPSCs adhere to the cell culture vessel, they may be cultured for about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days.

[0092] An iPSC colony may be a mass of cells grown by adherent culture of a single iPSC cell. An iPSC colony may be cells that have the same genetic information.

[0093] Second, the method includes differentiating the iPSC colonies into mesodermal cells to generate mesodermal cells.

[0094] In this case, the number of colonies on the day of initiation of differentiation from iPSC colonies into mesodermal cells may be 1 to 20. Furthermore, the total number of iPSC cells on the day of initiation of differentiation from iPSC colonies into mesodermal cells is 1 cm. 2 0.5-1×10 per cell culture vessel area 6 In this case, the total number of stem cells on the day of initiation of differentiation is the same as above. Preferably, the total number of iPSC cells on the day of initiation of differentiation from an iPSC colony to a mesoderm cell is 1 cm 2 1 x 10 per cell culture vessel area 3 ~1×10 6 It may also be a cell.

[0095] As used herein, the term "mesodermal cells" refers to a stratified population of cells that lie between the ectoderm and endoderm and are generated during blastocyst formation and are found in animals higher than flatworms. Mesodermal cells can differentiate into blood cells; vascular endothelial cells; muscle, including smooth and cardiac muscle; and connective tissues such as bone, cartilage, and fat.

[0096] In this case, the iPSCs can have a size of about 10 μm to about 3,000 μm per colony.

[0097] Specifically, iPSCs may have a size of about 10 μm to about 3,000 μm, about 50 μm to about 2,500 μm, about 100 μm to about 2,000 μm, about 300 μm to about 1,200 μm, about 400 μm to about 800 μm, or about 500 μm to about 700 μm per colony. Preferably, iPSCs may have a size of about 300 μm to about 1,200 μm per colony, but are not limited thereto.

[0098] Additionally, the iPSC colonies may be cultured in serum-free medium. The iPSC colonies may be cultured in a medium capable of culturing stem cells.

[0099] The iPSC colonies may be cultured in a medium containing at least one selected from the group consisting of glycogen synthase kinase-3 (GSK3) inhibitor, activin-like receptor kinase 5 (ALK5) inhibitor, bone morphogenetic protein 4 (BMP4), stem cell factor (SCF), and vascular endothelial growth factor (VEGF). The GSK3 inhibitor may be selected from the group consisting of CHIR-99021, SB216763, AT7519, CHIR-98014, TWS119, tideglusib, SB415286, and BIO (6-bromoindirubin-3-oxime, 6-bromoindirubin-3'-oxime). The ALK5 inhibitor may be selected from the group consisting of SB431542, galunisertib, LY2109761, SB525334, SB505124, GW788388, and LY364947.

[0100] iPSC colonies may be cultured in medium containing CHIR-99021, BMP4 and VEGF, and then cultured in medium containing SB431542, SCF and VEGF.iPSC colonies may be cultured in medium containing CHIR-99021, SB431542, BMP4, SCF and VEGF.

[0101] The iPSC colonies may be cultured in the above medium for about 1 hour to about 2 weeks, about 12 hours to about 2 weeks, about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days. Preferably, the iPSC colonies may be cultured for about 2 days to about 4 days, but are not limited thereto.

[0102] The mesodermal cells may be mesodermal stem cells.

[0103] Third, the method includes differentiating the mesodermal cells into hematopoietic stem cells to generate hematopoietic stem cells.

[0104] As used herein, the term "hematopoietic stem cells (HSCs)" may be used interchangeably with "hematopoietic precursor cells" or "hematopoietic progenitor cells" and refers to progenitor cells that can differentiate into hematopoietic cells. That is, hematopoietic stem cells (HSCs)" are pluripotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages), granulocytes (neutrophils, basophils, eosinophils, and mast cells), erythrocytes, megakaryocytes / platelets, and dendritic cells), and lymphoid (T cells, B cells, natural killer cells) (see [Doulatov et al., 2012; Notta et al., 2015]). Hematopoietic stem cells may express CD34 and CD133 and may be negative for CD38 expression. Hematopoietic progenitor cells may include CD34+ / CD45+ hematopoietic progenitor cells and CD34+ / CD45+ / CD43+ hematopoietic progenitor cells.

[0105] In this case, the mesodermal cells may be cultured in a serum-free medium. Additionally, the mesodermal cells may be cultured in a medium containing STF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand), or a combination thereof.

[0106] The mesodermal cells may be cultured for about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days. Preferably, the mesodermal cells may be cultured for about 2 days to about 8 days, but are not limited thereto.

[0107] Cells differentiated from mesodermal cells into hematopoietic stem cells may express CD34, in which case the CD34 expression rate may be about 80% or more of the total cells induced to differentiate without a separate sorting step to select only CD34 expressing cells. Specifically, the CD34 expression rate of cells differentiated from mesodermal cells to hematopoietic stem cells may be about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more. Preferably, the CD34 expression rate of cells differentiated from mesodermal cells to hematopoietic stem cells may be about 85% or more.

[0108] The above-mentioned "CD34" is a marker protein for hematopoietic stem cells and is involved in cell-cell adhesion. The sorting step for selecting only CD34-expressing cells can be carried out by flow cytometry or a kit.

[0109] Fourth, the method includes differentiating the hematopoietic stem cells into natural killer cells to generate the natural killer cells.

[0110] In this case, the hematopoietic stem cells may be cultured in a serum-free medium. Additionally, the hematopoietic stem cells may be cultured in a medium containing STF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand), or a combination thereof.

[0111] The hematopoietic stem cells may be cultured in a medium comprising IL-7, IL-15, or a combination thereof.The hematopoietic stem cells may be cultured in a medium comprising STF, FLT3L, IL-7, IL-15, or a combination thereof.

[0112] Hematopoietic stem cells may be cultured for about 1 day to about 3 months, about 5 days to about 2 months, about 1 week to about 2 months, about 2 weeks to about 2 months, about 3 weeks to about 2 months, about 1 month to about 2 months, about 1 month to about 7 weeks, about 1 month to about 6 weeks, about 1 month to about 5 weeks, or about 5 weeks to about 6 weeks (e.g., about 36 days). Preferably, hematopoietic stem cells may be cultured for about 1 day to about 26 days, or about 1 day to about 48 days, but are not limited thereto.

[0113] In the present invention, the hematopoietic stem cells may be co-cultured with autologous feeder cells.

[0114] As used herein, the term "feeder cells" refers to cells that are co-cultured with cells of a second type and provide growth factors and nutrients that allow the growth of the cells of the second type. The feeder cells may optionally be derived from a species similar or heterologous to the cells they support. For example, certain types of human cells, including stem cells, may be supported by cultures of mouse fetal fibroblasts and non-apoptotic mouse fetal fibroblasts. The feeder cells may be human-derived feeder cells, such as human dermal fibroblasts or human embryonic stem cells. In this case, the feeder cells may be inactivated by treatment with antimitotic agents such as mitomycin or irradiation. Through such inactivation, when co-cultured with cells of a second type, the feeder cells produce and secrete cellular metabolites in a state of arrested cell division, thereby regulating the differentiation and growth of the cells of the second type.

[0115] In this case, in the present invention, the feeder cells may be autologous feeder cells.

[0116] In one embodiment, the autologous feeder cells of the present invention may be cells that play a role in regulating the differentiation and growth of hematopoietic stem cells into natural killer cells. In the present invention, the feeder cells may be cells that play a role in regulating the proliferation and activity of differentiated natural killer cells.

[0117] The above-mentioned "autologous feeder cells" of the present invention include cells induced to differentiate from iPSCs into mesodermal cells. Preferably, the above-mentioned "autologous feeder cells" may include, but are not limited to, all cells that are not differentiated into hematopoietic stem cells and adhere to the container among the cells induced to differentiate into hematopoietic stem cells together in the iPSC colony.

[0118] In this case, induction of differentiation of iPSCs into natural killer cells may be performed in vitro.

[0119] The differentiation of induced pluripotent stem cells into natural killer cells may be carried out by culturing for about 26 days. Specifically, the differentiation of iPSCs into natural killer cells may be carried out by culturing for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, or 26 days.

[0120] Furthermore, in the present invention, the natural killer cells derived from the induced pluripotent stem cells may be further cultured for 21 days after differentiation is completed. Specifically, the natural killer cells may be further cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days.

[0121] Furthermore, the present invention may further involve large scale culture expansion with natural killer cells induced to differentiate from the induced pluripotent stem cells.

[0122] In this case, when the natural killer cells induced to differentiate from the induced pluripotent stem cells are cultured in a medium containing STF, FLT3L, IL-7, IL-15, IL-2, or a combination thereof, the natural killer cells may be cultured in a medium containing β-mercaptoethanol, sodium selenite, ethanolamine, L-ascorbic acid, or a combination thereof. In this case, the natural killer cells induced to differentiate from the induced pluripotent stem cells may be cultured for about 1 day to about 9 weeks, about 1 day to about 8 weeks, about 1 day to about 7 weeks, about 1 day to about 6 weeks, about 1 day to about 5 weeks, about 1 day to about 4 weeks, or about 1 day to about 3 weeks for mass culture.

[0123] In the present invention, when natural killer cells induced to differentiate from induced pluripotent stem cells are cultured for mass expansion, the natural killer cells may be co-cultured with feeder cells, in which case the feeder cells are the same as those described above.

[0124] In this case, large scale culture expansion on natural killer cells derived from induced pluripotent stem cells may be performed in vitro.

[0125] In one embodiment of the present invention, natural killer cells derived from induced pluripotent stem cells may not express TRA-1-60 and SSEA4, which are marker proteins for induced pluripotent stem cells. TRA-1-60 and SSEA4 are marker proteins for stem cells, and the stem cells are the same as those described above.

[0126] In one embodiment of the present invention, the natural killer cells exhibited killing ability against blood cancer cell lines (Figures 26, 31, 34b, 39e, and Table 10). Furthermore, it was confirmed that the natural killer cells activated immune cells more than the natural killer cells obtained from blood, and the expression of the receptor for the natural killer cell activation inhibitory protein expressed in tumor cells was reduced (Figures 36, 37, 38, Tables 13, and 14). Furthermore, through this, it can be seen that the natural killer cells of the present invention are newly isolated natural killer cells that have high natural killer cell activation and excellent killing ability against tumor cells compared to natural killer cells cultured by conventional known methods or natural killer cells isolated from humans.

[0127] Cell therapy agent comprising natural killer cells and pharmaceutical composition comprising the same In another aspect of the present invention, there is provided a cellular therapy comprising the natural killer cells of the present invention as an active ingredient.

[0128] In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a disease, comprising the natural killer cells of the present invention as an active ingredient.

[0129] As used herein, the term "cellular therapy" refers to drugs used with cells and tissues prepared through isolation, culture, and special production from a subject for treatment, diagnosis, and prevention (US FDA regulation). Cellular therapy refers to drugs used through a series of actions such as expanding or selecting live autologous, allogeneic, or xenogeneic cells ex vivo, or modifying the biological properties of cells in other ways to restore the function of cells or tissues for treatment, diagnosis, and prevention. Cellular therapy is largely classified into somatic cell therapy and stem cell therapy depending on the degree of cell differentiation. In the present invention, the cellular therapy may be a somatic cell therapy.

[0130] The pharmaceutical compositions of the present invention may be used for the prevention and treatment of infectious diseases, cancer, neurological diseases, autoimmune diseases, and the like.

[0131] The infectious disease may be an infectious disease caused by hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, influenza, and the like.

[0132] The cancer may be liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, brain cancer, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, lymphoma, and the like.

[0133] The "neurological disorder" may be a neurological disorder caused by nerve damage or abnormal nerves, or a degenerative brain disease, where the nerve damage may be a transient conduction disorder, an axotomy, or a neurotmesis.

[0134] The neurological disease caused by nerve damage or abnormal nerves may be a neurological disease caused by nerve damage or abnormal nerves in the central nervous system, or a neurological disease caused by nerve damage or abnormal nerves in the peripheral nervous system.

[0135] The neurological disease caused by nerve damage or abnormal nerves in the central nervous system may be organic disease and functional disorder in the central nervous system, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Lewy body disease, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, spinocerebellar degeneration, cerebellar atrophy, post-traumatic stress disorder, amnesia, vascular dementia, and cerebral infarction.

[0136] Neurological diseases caused by nerve damage or abnormal nerves in the peripheral nervous system may be peripheral neuropathy, diabetic neuropathy, peripheral neuropathic pain, peripheral neuropathy caused by anti-cancer chemotherapy, complex regional pain syndrome, optic neuropathy, mononeuropathy, mononeuritis multiplex (mononeuropathy multiplex), polyneuropathy, Guillain-Barre syndrome (acute inflammatory demyelinating polyneuropathy), chronic inflammatory demyelinating polyneuropathy, hereditary neuropathy, plexopathy, glaucoma, macular degeneration, amyotrophic lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, poliomyelitis, post-polio syndrome, stiff-person syndrome, Isaacs syndrome, myasthenia gravis, neonatal myasthenia gravis, botulism, Eaton-Lambert syndrome, thoracic outlet syndrome, Charcot-Marie-Tooth disease, and spinal muscular atrophy, etc.

[0137] The above-mentioned "degenerative brain disease" refers to a degenerative disease that occurs in the brain with age. Degenerative brain disease may include all diseases caused by the death of brain nerve cells, problems in the formation or function of synapses that transmit information between brain nerve cells, or abnormal increase or decrease in electrical activity of brain nerves. Degenerative brain disease may include, but is not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down's syndrome, amyloid stroke, systemic amyloid disease, Dutch-type amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedreich's ataxia, Machado-Joseph disease, Lewy body disease, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

[0138] As used herein, the term "autoimmune disease" is a general term that refers to diseases caused by antigen-antibody reaction due to antibodies produced against one's own biological substances or body tissues. Autoimmune diseases may include, but are not limited to, Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's disease, Guillain-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic sclerosis, asthma, ulcerative colitis, and the like.

[0139] As used herein, the term "prevention" refers to any measure in which the administration of a pharmaceutical composition inhibits or delays the onset of a geriatric disease. The term "treatment" refers to any measure in which the symptoms of a disease are improved or beneficially altered by the administration of a pharmaceutical composition.

[0140] In the pharmaceutical composition of the present invention, the natural killer cells may be contained in any amount (effective amount) depending on the purpose, formulation, mixing purpose, and the like, so long as they can exhibit activity. The pharmaceutical composition of the present invention contains 1×10 2 ~1×10 16 , 1×10 2 ~1×10 15 , 1×10 2 ~1×10 14 , or 1 × 10 2 ~1×10 13 Preferably, the pharmaceutical composition comprises 2×10 activated natural killer cells. 2 ~2×10 11 The antibody may include activated natural killer cells.

[0141] Here, the term "effective amount" refers to the amount of active ingredient capable of inducing an anti-aging effect. Such an effective amount can be determined experimentally within the normal capabilities of a person skilled in the art.

[0142] The compositions of the present invention may be used unfrozen or may be frozen for later use. If frozen, standard cryopreservatives (e.g., DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, id erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, Epilife® Cell Freezing Medium (Cascade Biologics)) may be added to the cells prior to freezing.

[0143] A pharmaceutical composition containing the natural killer cells of the present invention as an active ingredient may be formulated by further including a pharma- ceutically acceptable carrier.

[0144] As used herein, the term "pharmaceutical acceptable carrier" refers to a carrier or diluent that does not significantly stimulate an organism and does not suppress the biological activity and properties of the administered component. The pharmaceutical composition of the present invention may be applied as a cell therapy drug, and pharmaceutical acceptable carriers that may be included in the cell therapy drug, such as buffers, preservatives, analgesics, solubilizers, isotonicity agents, stabilizers, bases, excipients, lubricants, and the like, may be used without limitation as long as they are known in the art. The pharmaceutical composition of the present invention may be prepared in various formulations according to commonly used techniques.

[0145] The pharmaceutical composition of the present invention may be administered in a pharmacologic effective amount and may be administered through any route as long as the route can induce migration to the affected area. Here, "administration" means introducing a predetermined substance into a subject by a suitable method. In some cases, the pharmaceutical composition of the present invention is loaded into a medium that includes a means for directing natural killer cells to the lesion.

[0146] Therefore, the pharmaceutical composition of the present invention may be administered through several routes, including topical (including buccal, sublingual, transdermal and intraocular), parenteral (including subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular and intracerebrospinal fluid) or transdermal, preferably directly to the site of the disease. In one embodiment, the natural killer cells may be administered to a subject suspended in a suitable diluent, which is used to protect and maintain the cells when injected into the target tissue, facilitating their use. The diluent may include buffers such as saline, phosphate buffer, and HBSS, cerebrospinal fluid, and the like. Furthermore, the composition may be administered by any device that transfers the active agent to the target cells.

[0147] The preferred mode of administration and formulation is injection. Injections may be prepared using aqueous solutions such as physiological saline, Ringer's solution, Hank's solution or sterile aqueous solution, vegetable oils such as olive oil, higher fatty acid esters such as ethyl oleate, and non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol or glycerin, and the like. For mucosal penetration, non-penetrating agents known in the art suitable for the barrier to be crossed may be used, and may further include pharmaceutical carriers such as ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA, and the like, as stabilizers to prevent deterioration, as well as preservatives to inhibit microbial growth, such as emulsifiers, buffers to adjust pH, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzyl alcohol. Methods for formulating pharmaceutical compositions are known in the art, and specific reference may be made to the literature [Remington's Pharmaceutical Sciences (19th Edition, 1995)], etc., which are considered to be part of this specification.

[0148] As used herein, the term "pharmaceutical effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio that is applicable to medical treatment and does not cause side effects.Effective dose level can be easily determined by those skilled in the art based on factors including the patient's sex, age, weight, health condition, type and severity of disease, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.

[0149] The preferred dosage of the pharmaceutical composition of the present invention is about 1×10 / kg to about 1×10 / kg per day on a cell basis, depending on the condition, weight, sex, and age of the patient, the severity of the patient, and the route of administration. 12 / kg, approx. 1×10 / kg to approx. 1×10 11 / kg, or about 1×10 / kg to about 1×10 10 / kg is also acceptable.

[0150] However, the dosage may be formulated in various ways depending on factors such as the formulation method, mode of administration, age, body weight, sex, pathology, diet, administration time, administration route, excretion rate, and response sensitivity of the patient, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The number of administrations may be once or twice or more within the range of clinically acceptable side effects. The pharmaceutical composition of the present invention may be administered 1 to 3 times / day, 1 to 3 times / week, or 1 to 10 times / month. For example, the pharmaceutical composition of the present invention may be administered 1 to 9 times / month, 1 to 8 times / month, 1 to 7 times / month, 1 to 6 times / month, 1 to 5 times / month, 1 to 4 times / month, 1 to 3 times / month, or 1 to 2 times / month. With respect to the administration site, the pharmaceutical composition of the present invention may be administered to one site or two or more sites. In animals other than humans, the pharmaceutical composition of the present invention may be administered in the same dosage as in humans per kg or per subject, or the pharmaceutical composition of the present invention may be administered in an amount converted from the dosage above, for example, by the volume ratio (e.g., average value) of organs (e.g., heart, etc.) between the target animal and humans. Such dosages should not be construed in any way as limiting the scope of the present invention.

[0151] As used herein, the term "subject" refers to a subject that has or may develop a disease to which the composition of the present invention may be applied (prescribed), and may be a mammal, such as a rat, mouse, or livestock, including a human. Preferably, the subject may be a human, but is not limited thereto.

[0152] The pharmaceutical composition of the present invention may further contain, if necessary, a known substance that exhibits preventive or therapeutic effects against a disease.

[0153] The pharmaceutical composition of the present invention may further comprise the above-mentioned substance that increases the activity of natural killer cells. The pharmaceutical composition of the present invention may be administered in combination with a substance conventionally known for preventing or treating a disease. In this case, the natural killer cells, the substance exhibiting a preventive or therapeutic effect against a disease, and / or the substance that increases the activity of natural killer cells may be administered simultaneously or sequentially.

[0154] In another aspect of the present invention, there is provided a use of natural killer cells or a pharmaceutical composition comprising natural killer cells as an active ingredient for the prevention and treatment of a disease, wherein the natural killer cells, pharmaceutical composition, disease, prevention and treatment are the same as above.

[0155] In another aspect of the present invention, there is provided a method for treating a disease, comprising administering natural killer cells or a pharmaceutical composition comprising natural killer cells as an active ingredient to a subject, wherein the natural killer cells, pharmaceutical composition, disease, administration and treatment are as described above.

[0156] The subject may be any animal, including humans, such as rats, mice, or livestock, that suffers from or may develop a disease. Preferably, the subject is a human.

[0157] The dosage is about 1 × 10 / kg to about 1 × 10 per day on a cell basis, depending on the condition, weight, sex, and age of the patient, the severity of the patient, and the route of administration. 12 / kg, approx. 1×10 / kg to approx. 1×10 11 / kg, or about 1×10 / kg to about 1×10 10 However, the dosage may be formulated in various ways depending on factors such as the formulation method, the mode of administration, the age, weight, sex, pathology, diet, administration time, administration route, excretion rate, and response sensitivity of the patient, and a person skilled in the art can appropriately adjust the dosage by considering these factors.

[0158] Methods for Producing Natural Killer Cells In another aspect of the invention, a method for producing natural killer cells is provided comprising the steps of: 2 The method includes the steps of seeding induced pluripotent stem cells as iPSCs or iPSC spheroids into a cell culture vessel and performing adhesion culture to produce iPSC colonies so that 0.5 to 20 colonies are formed per area of ​​the cell culture vessel; differentiating the iPSC colonies into mesodermal cells to produce mesodermal cells; differentiating the mesodermal cells into hematopoietic stem cells to produce hematopoietic stem cells; and differentiating the hematopoietic stem cells into natural killer cells to produce natural killer cells.

[0159] In this case, the induced pluripotent stem cells may be cultured by adherent culture methods, or may be seeded as single cells or spheroids and subjected to adherent culture, the number of cells contained in the spheroids being the same as above.

[0160] Furthermore, the number of seeded iPSCs or iPSC spheroids was determined based on the number of cells per 1 cm 2 The number of iPSCs or iPSC spheroids seeded may be 0.5 to 20 per area. The iPSCs or iPSC spheroids seeded are the same as above.

[0161] Additionally, the number of cells seeded can be calculated in different ways. The number of cells present per ml of culture can be calculated per cm by using the method for calculating the number of cells seeded. 2 The amount of the cells per well can be calculated.

[0162] Furthermore, the method for producing natural killer cells of the present invention may further comprise mass culture expansion of natural killer cells differentiated from induced pluripotent stem cells.

[0163] In this case, the induced pluripotent stem cells, differentiation, natural killer cells, and culture are the same as above. In addition, the number of iPSCs seeded and the size of 1 cm 2 The methods for calculating the number of cells seeded per cell culture vessel area in different ways are the same as above.

[0164] iPSCs may be cultured in a medium containing a ROCK inhibitor. In this case, the ROCK inhibitor is the same as above. iPSCs may be cultured in a medium containing a ROCK inhibitor for about 10 minutes to about 1 week, about 30 minutes to about 6 days, about 1 hour to about 5 days, about 2 hours to about 4 days, about 3 hours to about 3 days, about 6 hours to about 2 days, about 12 hours to about 2 days, or about 18 hours to about 1 day. Furthermore, iPSCs may be cultured by an adherent culture method. Single-cell iPSCs may be cultured for about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days after attachment to a cell culture vessel.

[0165] An iPSC colony may be a mass of cells grown by adherent culture of a single iPSC cell. An iPSC colony may be cells that have the same genetic information.

[0166] The iPSC colonies may be differentiated into mesodermal cells to produce mesodermal cells. The total number of induced pluripotent stem cells and mesodermal cells on the day of initiation of differentiation are the same as above. In this case, the total number of iPSC stem cells on the day of initiation of differentiation of the iPSC colonies into mesodermal cells is 1 cm 2 0.5-1×10 per cell culture vessel area 6The iPSCs may be cells. In this case, the iPSCs may have a size of about 10 μm to about 3,000 μm per colony. Specifically, the iPSCs may have a size of about 10 μm to about 3,000 μm, about 50 μm to about 2,500 μm, about 100 μm to about 2,000 μm, about 300 μm to about 1,200 μm, about 400 μm to about 800 μm, or about 500 μm to about 700 μm per colony. Preferably, the iPSCs may have a size of about 300 μm to about 1,200 μm per colony, but is not limited thereto.

[0167] Furthermore, the iPSC colony may be cultured in a serum-free medium. The iPSC colony may be cultured in a medium capable of culturing stem cells. Furthermore, the iPSC colony may be cultured in a medium comprising at least one selected from the group consisting of GSK3 inhibitor, ALK5 inhibitor, BMP4, SCF, and VEGF. The GSK3 inhibitor and the ALK5 inhibitor are the same as above.

[0168] The iPSC colony may be cultured in a medium containing CHIR-99021, BMP4, and VEGF, and then cultured in a medium containing SB431542, SCF, and VEGF. The iPSC colony may be cultured in a medium containing CHIR-99021, SB431542, BMP4, SCF, and VEGF. In this case, the iPSC colony may be cultured in the above medium for about 1 hour to about 2 weeks, about 12 hours to about 2 weeks, about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days. Preferably, the iPSC colony may be cultured for about 2 days to about 4 days, but is not limited thereto.

[0169] The mesodermal cells may be mesodermal stem cells.

[0170] The mesodermal cells can be differentiated to produce hematopoietic stem cells, where the hematopoietic stem cells are the same as described above.

[0171] The mesodermal cells may be cultured in a serum-free medium or in a medium containing STF, FLT3L, or a combination thereof. In this case, the mesodermal cells may be cultured for about 1 day to about 2 weeks, about 2 days to about 13 days, about 3 days to about 12 days, about 4 days to about 11 days, about 5 days to about 10 days, about 6 days to about 9 days, or about 7 days to about 8 days. Preferably, the mesodermal cells may be cultured for about 2 days to about 8 days, but are not limited thereto.

[0172] Cells differentiated from mesodermal cells to hematopoietic stem cells may express CD34, in which case the CD34 expression rate may be about 80% or more of the total cells induced to differentiate without a separate sorting step to select only CD34 expressing cells. Specifically, the CD34 expression rate of all cells differentiated from mesodermal cells to hematopoietic stem cells may be about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more. Preferably, the CD34 expression rate of all cells differentiated from mesodermal cells to hematopoietic stem cells may be about 85% or more.

[0173] The hematopoietic stem cells may be cultured in a medium comprising IL-7, IL-15, or a combination thereof.The hematopoietic stem cells may be cultured in a medium comprising STF, FLT3L, IL-7, IL-15, or a combination thereof.

[0174] Hematopoietic stem cells may be cultured for about 1 day to about 3 months, about 5 days to about 2 months, about 1 week to about 2 months, about 2 weeks to about 2 months, about 3 weeks to about 2 months, about 1 month to about 2 months, about 1 month to about 7 weeks, about 1 month to about 6 weeks, about 1 month to about 5 weeks, or about 5 weeks to about 6 weeks (e.g., about 36 days). Preferably, hematopoietic stem cells may be cultured for about 1 day to about 26 days, or about 1 day to about 48 days, but are not limited thereto.

[0175] In the present invention, the hematopoietic stem cells may be co-cultured with autologous feeder cells.

[0176] The feeder cells are the same as those described above. In this case, in the present invention, the feeder cells may be autologous feeder cells.

[0177] In one embodiment, the autologous feeder cells of the present invention may be cells that play a role in regulating the differentiation and growth of hematopoietic stem cells into natural killer cells. In the present invention, the feeder cells may be cells that play a role in regulating the proliferation and activity of differentiated natural killer cells.

[0178] The above-mentioned "autologous feeder cells" of the present invention include cells induced to differentiate from iPSCs into mesodermal cells. Preferably, the above-mentioned "autologous feeder cells" may include, but are not limited to, all cells that are not differentiated into hematopoietic stem cells and adhere to the container among the cells induced to differentiate into hematopoietic stem cells together in the iPSC colony.

[0179] The method may be performed in vitro.

[0180] The medium may be a known medium used in cell culture or a modified medium thereof.

[0181] The method for producing natural killer cells of the present invention may further comprise large scale culture expansion of natural killer cells that are induced to differentiate.

[0182] In this case, during mass culture, the natural killer cells induced to differentiate from the induced pluripotent stem cells may be cultured in a medium containing STF, FLT3L, IL-7, IL-15, IL-2, or a combination thereof. Furthermore, the natural killer cells induced to differentiate from the induced pluripotent stem cells may be cultured in a medium further containing β-mercaptoethanol, sodium selenite, ethanolamine, L-ascorbic acid, or a combination thereof. In this case, the mass culture may be performed for about 1 day to about 4 weeks, or about 1 day to about 3 weeks.

[0183] In the present invention, when natural killer cells induced to differentiate from induced pluripotent stem cells are cultured in large quantities, the natural killer cells may be co-cultured with feeder cells, in which case the feeder cells are the same as those described above.

[0184] In this case, large scale culture expansion of natural killer cells derived from induced pluripotent stem cells may be performed in vitro.

[0185] The natural killer cells produced by the above method may express any one of the proteins selected from the group consisting of CD25, CD9, CD80, and combinations thereof. In addition, the natural killer cells may further express any one of the proteins selected from the group consisting of CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, CD210, and combinations thereof.

[0186] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, or about 95% or more of the natural killer cell population generated by the methods of the present invention may express CD25.

[0187] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, or about 97% or more of the natural killer cell population generated by the methods of the present invention may express CD80.

[0188] Specifically, about 10% or more, about 20% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, or about 34% or more of the natural killer cell population generated by the methods of the present invention may express CD9.

[0189] Specifically, about 5% or more, about 6% or more, or about 7% or more of a natural killer cell population generated by the methods of the present invention may express CD1d.

[0190] Specifically, about 20% or more, about 30% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, or about 45% or more of a natural killer cell population generated by the methods of the present invention may express CD30.

[0191] Specifically, about 20% or more, about 30% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, about 45% or more, or about 46% or more of a natural killer cell population generated by the methods of the present invention may express CD87.

[0192] Specifically, about 10% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, or about 26% or more of a natural killer cell population generated by the methods of the present invention may express CDw327.

[0193] Specifically, about 10% or more, about 11% or more, about 12% or more, about 13% or more, or about 14% or more of a natural killer cell population generated by the methods of the present invention may express CD33.

[0194] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the natural killer cell population generated by the methods of the present invention may express CD49a.

[0195] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, or about 94% or more of the natural killer cell population generated by the methods of the present invention may express CD49c.

[0196] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, or about 96% or more of the natural killer cell population generated by the methods of the present invention may express CD49e.

[0197] Specifically, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, or about 15% or more of the natural killer cell population generated by the methods of the present invention may express CD49f.

[0198] Specifically, about 50% or more, about 60% or more, about 70% or more, about 74% or more, about 75% or more, about 76% or more, about 77% or more, about 78% or more, about 79% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, or about 84% or more of the natural killer cell population generated by the methods of the present invention may express CD66(a / b / c / d / e).

[0199] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, or about 96% or more of the natural killer cell population generated by the methods of the present invention may express CD71.

[0200] Specifically, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, about 35% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, or about 40% or more of the natural killer cell population generated by the methods of the present invention may express CD83.

[0201] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, or about 95% or more of the natural killer cell population generated by the methods of the present invention may express CDw93.

[0202] Specifically, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 31% or more, or about 32% or more of the natural killer cell population generated by the methods of the present invention may express CD106.

[0203] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, or about 94% or more of the natural killer cell population generated by the methods of the present invention may express CD108.

[0204] Specifically, about 20% or more, about 30% or more, about 35% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, about 40% or more, about 41% or more, or about 42% or more of the natural killer cell population generated by the methods of the present invention may express CD114.

[0205] Specifically, about 10% or more, about 20% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, or about 35% or more of the natural killer cell population generated by the methods of the present invention may express CD123.

[0206] Specifically, about 5% or more, about 6% or more, about 7% or more, about 8% or more, or about 9% or more of a natural killer cell population generated by the methods of the present invention may express CD124.

[0207] Specifically, about 50% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, or about 65% or more of the natural killer cell population generated by the methods of the present invention may express CD146.

[0208] Specifically, about 10% or more, about 20% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 31% or more, or about 32% or more of the natural killer cell population generated by the methods of the present invention may express CD150.

[0209] Specifically, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, or about 97% or more of the natural killer cell population generated by the methods of the present invention may express CD164.

[0210] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, or about 95% or more of the natural killer cell population generated by the methods of the present invention may express CD196.

[0211] Specifically, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, or about 10% or more of the natural killer cell population generated by the methods of the present invention may express CD210.

[0212] The expression levels of the CD25, CD9, CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196 and CD210 proteins may be reduced by 5-fold or more compared to natural killer cells isolated from blood. Specifically, the expression levels may be increased by about at least 2-fold. Natural killer cells obtained from blood, CD25, CD9, CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, and CD210 are the same as above.

[0213] Natural killer cells may express any one of the natural killer cell marker proteins selected from the group consisting of CD56, CD45, CD16, NKp30, NKp44, NKG2D, DNAM1, and combinations thereof, and may not express the T cell marker protein, CD3, or may express CD3 at a lower level compared to T cells.

[0214] Specifically, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, or about 96% or more of the natural killer cell population generated by the methods of the present invention may express CD56, CD45, NKp30 or DNAM1.

[0215] Specifically, about 40% or more, about 50% or more, about 51% or more, about 52% or more, about 53% or more, about 54% or more, about 55% or more, about 56% or more, about 57% or more, or about 58% or more of the natural killer cell population generated by the methods of the present invention may express CD16.

[0216] Specifically, about 50% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, about 64% or more, about 65% or more, about 66% or more, about 67% or more, about 68% or more, about 69% or more, about 70% or more, about 71% or more, or about 72% or more of a natural killer cell population generated by the methods of the present invention may express NKp44.

[0217] Specifically, about 70% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, or about 89% or more of the natural killer cell population generated by the methods of the present invention may express NKD2D.

[0218] Furthermore, about 90% or more, about 9% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, or about 99.4% or more of the natural killer cell population generated by the methods of the present invention may not express CD3.

[0219] Furthermore, the natural killer cells according to the method of the present invention may further increase the expression of any one gene selected from the group consisting of GZMB, CD226, RASGRP1, CD107a, CCR9, CD40LG, CD300c, and combinations thereof, compared to natural killer cells isolated from blood. The above GZMB, CD226, RASGRP1, CD107a, CCR9, CD40LG, and CD300c are the same as above.

[0220] The natural killer cells according to the present invention may be characterized by increased secretion of any one of the proteins selected from the group consisting of FLT3L, MIP1 alpha / beta, PF4, IGFBP2, IGFBP3, MMP9, and combinations thereof, compared to natural killer cells isolated from blood. In this case, the secretion of the above proteins may be increased by at least two-fold compared to natural killer cells isolated from blood. The natural killer cells obtained from blood, FLT3L, MIP1 alpha / beta, PF4, IGFBP2, IGFBP3, and MMP9 are the same as above.

[0221] The natural killer cells according to the present invention may not further express any one of the proteins selected from the group consisting of CD205, CD158b, CD243, γδ TCR, NKB1, CD158a, CDw328, TIM3, TIGIT, and combinations thereof, or may express any one of the proteins at a lower level compared to natural killer cells obtained from blood. Natural killer cells obtained from blood, CD205, CD158b, CD243, γδ TCR, NKB1, CD158a, CDw328, TIM3, and TIGIT are the same as above.

[0222] Specifically, about 90% or more, about 95% or more, about 96% or more, or about 97% or more, about 97.5% or more, about 97.6% or more, about 97.7% or more, about 97.8% or more, or about 97.9% or more of the natural killer cell population generated by the methods of the present invention may not express CD205.

[0223] Furthermore, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 97.1% or more, about 97.2% or more, about 97.3% or more, or about 97.4% or more of the natural killer cell population generated by the methods of the present invention may not express CD158b.

[0224] Furthermore, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, about 99.4% or more, about 99.5% or more, about 99.6% or more, or about 99.7% or more of the natural killer cell population generated by the methods of the present invention may not express CD243.

[0225] Furthermore, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, about 99.4% or more, about 99.5% or more, about 99.6% or more, or about 99.7% or more of the natural killer cell population generated by the methods of the invention may not express γδ TCR.

[0226] Furthermore, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, about 99.4% or more, or about 99.5% or more of the natural killer cell population generated by the methods of the present invention may not express NKB1.

[0227] Furthermore, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.1% or more, about 99.2% or more, about 99.3% or more, about 99.4% or more, about 99.5% or more, or about 99.6% or more of the natural killer cell population generated by the methods of the present invention may not express CD158a.

[0228] Furthermore, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 98.1% or more, about 98.2% or more, about 98.3% or more, about 98.4% or more, about 98.5% or more, about 98.6% or more, or about 98.7% or more of the natural killer cell population generated by the methods of the present invention may not express CDw328.

[0229] Furthermore, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the natural killer cell population generated by the methods of the invention may not express TIM-3.

[0230] Furthermore, about 80% or more, about 85% or more, about 90% or more, about 93% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more of the natural killer cell population produced by the methods of the present invention may not express TIGIT.

[0231] The expression levels of the above CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM-3, and TIGIT proteins may be reduced to about half or less compared to natural killer cells obtained from blood. The natural killer cells obtained from blood are the same as those described above.

[0232] Typically, it was confirmed that when iPSCs were subjected to the third culture using Aggrewell, the size of the embryoid bodies was larger than that of the second culture (adherent culture) (Figure 44). Furthermore, it was confirmed that there was a difference in the size of the embryoid bodies depending on the batch, and that when single cells were obtained by treatment with degradative enzymes, the cell viability was reduced (Figure 45). Meanwhile, the method for producing natural killer cells according to the present invention may be simpler and more efficient than the third culture, because iPSCs were subjected to the second culture (adherent culture) and induced to differentiate.

[0233] Furthermore, when natural killer cells are produced from induced pluripotent stem cells by a conventional method, differentiation is induced from mesodermal cells to hematopoietic stem cells to obtain only CD34 positive cells (hematopoietic stem cells), which are then induced to differentiate into natural killer cells. In this case, the step of selecting only CD34 positive cells may be performed using flow cytometry or a commercially available sorting kit. In the case of cells subjected to the step of selecting CD34 positive cells by this method, it was confirmed that the cell yield was significantly decreased, cell death was increased, and the expression rate of natural killer cell marker protein (CD56) and activation marker protein (NKp30) in the selected CD34 positive cells was decreased (Figures 42a and 42b). Furthermore, it was confirmed that the cell proliferation rate was significantly decreased (Figure 42c). On the other hand, in one embodiment of the present invention, the method for producing natural killer cells of the present invention may induce differentiation into natural killer cells without performing a CD34 positive cell sorting step. Because the expression rate of CD34, a hematopoietic stem cell differentiation marker, at the intermediate stage of NK differentiation was as high as 95% or higher, thereby increasing cell yield and activity (FIG. 42d).

[0234] Furthermore, while the conventional method for producing natural killer cells requires a total differentiation induction period of 48 days, the method for producing natural killer cells according to the present invention requires a differentiation induction period of 26 days in one embodiment of the present invention. As a result of checking natural killer cell marker proteins (CD56, CD45, NKp44) and activation proteins (CD56, NKp30) in EiNK cells induced to differentiate for 26 days, the expression rate of NK marker proteins and activation proteins was confirmed to be 66% or higher. Meanwhile, the expression rate of iPSC marker proteins (TRA-1-60, SSEA4) was confirmed to be 0%, indicating that the method for producing natural killer cells according to the present invention is an efficient method for producing natural killer cells in a short period of time compared to existing methods (Figures 45 and 46).

[0235] Methods for culturing stem cells In one aspect of the invention, a method for culturing stem cells is provided, comprising: 2 The method includes the step of seeding stem cells as iPSCs or iPSC spheroids into a cell culture vessel such that 1-20 iPSC colonies are formed per area of ​​the cell culture vessel.

[0236] In this case, the stem cells may be induced pluripotent stem cells (iPSCs). Furthermore, in this case, the stem cells may be cultured by adherent culture. In this case, the stem cells may be seeded as spheroids and undergo adherent culture. The number of cells contained in the spheroids is the same as above.

[0237] Furthermore, the number of seeded iPSCs or iPSC spheroids was determined based on the number of cells per 1 cm 2 The number of iPSCs or iPSC spheroids seeded may be 0.5 to 20 per area. The iPSCs or iPSC spheroids seeded are the same as those described above.

[0238] Additionally, the number of cells seeded may be calculated in different ways. The number of cells present per ml of culture medium can be calculated per cm by using the method for calculating the number of cells seeded. 2 The number of cells per well may be determined.

[0239] The specific culture method is the same as above.

[0240] Methods for Differentiating Stem Cells In another aspect of the present invention, a method for differentiating stem cells is provided. Specifically, the method and the total number of stem cells on the day of starting differentiation of the stem cells are the same as above. Preferably, the total number of stem cells on the day of starting differentiation of the stem cells is 1 cm 2 1 x 10 per cell culture vessel area 3 ~1×10 6 The stem cells may be cells. The stem cells may be differentiated in various ways depending on the medium added. In one embodiment, the stem cells may be differentiated into mesodermal cells. The specific culture method is the same as above.

[0241] Methods for generating natural killer cells from iPSCs In another aspect of the present invention, a method for generating natural killer cells from iPSCs is provided, comprising: 2 The method includes the steps of: seeding induced pluripotent stem cells as iPSCs or iPSC spheroids into a cell culture vessel and performing adherent culture to produce iPSC colonies so that 0.5 to 20 colonies are formed per area of ​​the cell culture vessel; differentiating the iPSC colonies into mesodermal cells to produce mesodermal cells; differentiating the mesodermal cells into hematopoietic stem cells to produce hematopoietic stem cells; and differentiating the hematopoietic stem cells into natural killer cells to produce natural killer cells.

[0242] First, the method is 2 The method includes a step of seeding induced pluripotent stem cells (iPSCs) as iPSCs or iPSC spheroids into a cell culture vessel and performing adherent culture to produce iPSC colonies so that 0.5 to 20 colonies are formed per area of ​​the cell culture vessel. The number of cells seeded is the same as above.

[0243] Second, the method includes differentiating the iPSC colonies into mesodermal cells to generate mesodermal cells.

[0244] Third, the method includes differentiating the mesodermal cells into hematopoietic stem cells to generate hematopoietic stem cells.

[0245] Fourth, the method includes differentiating the hematopoietic stem cells into natural killer cells to generate the natural killer cells.

[0246] The specific methods and conditions are the same as above.

[0247] Method for culturing hematopoietic stem cells by co-culturing with autologous feeder cells - Patents.com In the present invention, the hematopoietic stem cells may be co-cultured with autologous feeder cells.

[0248] As used herein, the term "feeder cells" refers to cells that are co-cultured with cells of a second type and provide growth factors and nutrients that allow the growth of the cells of the second type. The feeder cells may optionally be derived from a species similar or heterologous to the cells they support. For example, certain types of human cells, including stem cells, may be supported by cultures of mouse fetal fibroblasts and non-apoptotic mouse fetal fibroblasts. The feeder cells may be human-derived feeder cells, such as human dermal fibroblasts or human embryonic stem cells. In this case, the feeder cells may be inactivated by treatment with antimitotic agents such as mitomycin or irradiation. Through such inactivation, when co-cultured with cells of a second type, the feeder cells produce and secrete cellular metabolites in a state of arrested cell division, thereby regulating the differentiation and growth of the cells of the second type.

[0249] In this case, in the present invention, the feeder cells may be autologous feeder cells.

[0250] The specific culture methods and conditions are the same as those described above.

[0251] Modes for carrying out the invention The present invention will now be described in more detail by the following examples, which are intended to illustrate one or more embodiments, and are not intended to limit the scope of the present invention.

[0252] I. Generation of natural killer cells Example 1. Method for generating natural killer cells from induced pluripotent stem cells A schematic diagram of the method for obtaining natural killer cells from induced pluripotent stem cells is as shown in Figure 1. Furthermore, the specific experimental method is as shown in Figures 2 and 19. The method can be roughly divided into the following steps: 1) generating induced pluripotent stem cell colonies (adherent cells) from induced pluripotent stem cells (adherent cells), 2) differentiating the induced pluripotent stem cells (adherent cells) into mesodermal cells (adherent cells), 3) differentiating the mesodermal cells (adherent cells) into hematopoietic stem cells (suspension cells) and autologous feeder cells (adherent cells), and 4) differentiating the hematopoietic stem cells (suspension cells) into natural killer cells while maintaining or removing the autologous feeder cells (adherent cells).

[0253] Example 1.1. Culturing induced pluripotent stem cells The CMC-009 induced pluripotent stem cell (iPSC) cell line used in this example was provided by the National Stem Cell Bank of the National Center for Stem Cell and Regenerative Medicine. The cell line was cultured in cell culture vessels coated with extracellular matrix iMatrix-511 (Nippi, 892012) under StemFit Basic04 culture medium (Ajinomoto, AJBASIC04).

[0254] Example 1.2. Formation of uniform induced pluripotent stem cell colonies from induced pluripotent stem cells Example 1.2.1. Formation of uniform induced pluripotent stem cell colonies from single induced pluripotent stem cells As a preliminary step for differentiation of human induced pluripotent stem cells into natural killer cells, the formation of induced pluripotent stem cell colonies (iPSC colonies) derived from single-cell induced pluripotent stem cells was induced.

[0255] Specifically, 1 cm 2Cell culture vessels for single cell iPSC attachment were prepared by diluting 1 μl of iMatrix-511 in 150 μl of DPBS (Cytiva, SH3002802) per cell culture vessel and applying it to the cell culture vessel for at least 1 h, then replacing it with StemFit Basic 04 culture medium containing 10 μM Y-27632. The iPSCs were treated with TypLE (Gibco, 12604013) to dissociate the iPSCs into single cells, and then plated on a 1 cm 2 0.5–20 single-cell iPSCs per culture vessel area were seeded into the prepared cell culture vessel.

[0256] The next day, the cell culture medium was replaced with fresh StemFit Basic 04 and cultured for another 9–12 days. When the iPSC colonies reached a diameter of 300–1,600 μm (average diameter 300–700 μm), the stem cell culture medium was removed and natural killer cell differentiation was initiated. During the process of generating induced pluripotent stem cell colonies, the cell culture medium was replaced every day.

[0257] Example 1.2.2. Formation of uniform induced pluripotent stem cell colonies from induced pluripotent stem cells in the form of spheroids As a preliminary step for differentiation of human induced pluripotent stem cells into natural killer cells, the formation of induced pluripotent stem cell colonies (iPSC colonies) was induced by seeding the induced pluripotent stem cells in the form of spheroids.

[0258] Specifically, 1 cm 2 1 μl of iMatrix-511 per cell culture vessel was diluted in 150 μl of DPBS (Cytiva, SH3002802) and applied to the cell culture vessel for at least 1 h, then replaced with StemFit Basic 04 culture medium containing 10 μM Y-27632 to prepare cell culture vessels on which iPSC spheroids were attached. iPSCs were prepared in the form of spheroids by culturing single iPSCs. This time, one spheroid contained about 1 to about 600 iPSCs. Then, 1 cm 20.5–20 iPSC spheroids per culture vessel area were seeded into the prepared cell culture vessel.

[0259] The next day, the cell culture medium was replaced with fresh StemFit Basic 04 and cultured for another 2-5 days. When the diameter of the induced pluripotent stem cell colonies (iPSC colonies) was 300-1,600 μm (average diameter 300-700 μm), the stem cell culture medium was removed and natural killer cell differentiation was initiated. During the process of generating induced pluripotent stem cell colonies, the cell culture medium was replaced every day.

[0260] Example 1.3. Generation of natural killer cells from induced pluripotent stem cell colonies Specifically, when the diameter of the induced pluripotent stem cell colonies (iPSC colonies) generated in Example 1.2 above reached 300-1,600 μm (average diameter 300-700 μm), the stem cell culture medium was removed and the cells were cultured for 2 days in serum-free stem cell culture medium Essential 8™ (Gibco, 1517001) further containing 2 μm CHIR-99021 (Peprotech, 2520691), 80 ng / ml BMP4 (R&D Systems, 314-BP / CF) and 80 ng / ml VEGF (R&D Systems, 293-VE / CF). After 2 days, the culture medium was removed and the cells were cultured in serum-free cell culture medium Essential 6™ (Gibco, 1516401) further containing 2 μM SB-431542 (Peprotech, 3014193), 50 ng / ml SCF (R&D Systems, 255-SC / CF) and 80 ng / ml VEGF to induce differentiation into mesodermal cells.

[0261] After 2 days, the culture medium was removed and the cells were cultured in serum-free hematopoietic stem cell culture medium Stemline II (Sigma, S0192) further containing 50ng / ml SCF and 50ng / ml FLT3L (R&D Systems, 308-FK / CF) to induce differentiation into hematopoietic stem cells. After 8 days, the culture medium was removed and replaced with Stemline II culture medium further containing 50ng / ml SCF, 50ng / ml FLT3L, IL-7 (R&D Systems, 207-IL-200 / CF) and IL-15 (R&D Systems, 247-ILB-500 / CF), and the cells were cultured for 36 days to finally induce differentiation into natural killer cells. This time, all culture media used for differentiation were half-replaced every 4 days.

[0262] Example 1.4. Generation of natural killer cells from iPSC colonies under conditions in which feeder cells are excluded Specifically, when the diameter of the induced pluripotent stem cell colony (iPSC colony) prepared in Example 1.2 above reached 700-1,200 μm, the stem cell culture medium was removed and the cells were cultured for 2 days in serum-free stem cell culture medium Essential 8™ further containing 2 μm CHIR-99021, 80 ng / ml BMP4 and 80 ng / ml VEGF. After 2 days, the culture medium was removed and the cells were cultured in serum-free cell culture medium Essential 6™ further containing 2 μm SB-431542, 50 ng / ml SCF and 80 ng / ml VEGF to induce differentiation into mesodermal cells.

[0263] After 2 days, the culture medium was removed, and the cells were cultured in serum-free hematopoietic stem cell culture medium Stemline II, which further contained 50ng / ml SCF and 50ng / ml FLT3L, to induce differentiation into hematopoietic stem cells. On the 12th day of differentiation, only floating cells in the cell culture vessel, excluding autologous feeder cells, were collected, transferred to a new cell culture vessel, and replaced with Stemline II culture medium, which further contained 50ng / ml SCF, 50ng / ml FLT3L, IL-7, and IL-15, and cultured for 36 days to finally induce differentiation into natural killer cells. This time, all culture media used for differentiation were half-replaced every 4 days.

[0264] Example 1.5. Verification of the morphology of natural killer cells differentiated from iPSC colonies by differentiation day During the differentiation of iPSCs into NK cells according to the methods of Examples 1.1 to 1.4, the morphological changes of the cells undergoing differentiation were observed using an optical microscope. Specifically, it was confirmed using an optical microscope (Olympus, Olympus-CKX53) that at each differentiation point, the morphology of the cells changed from pluripotent stem cells to mesodermal cells, hematopoietic stem cells, and then natural killer cells according to the differentiation stage.

[0265] The morphological changes of cells at each differentiation stage were confirmed using an optical microscope (Figure 3). In Figure 3, a photograph was taken on day 0 of an induced pluripotent stem cell colony (adherent cells) created from an induced pluripotent stem cell (adherent cells). In this case, the size of the colony in the photograph was 700 μm to 1,200 μm, and the colony of that size was differentiated into a mesoderm cell. In addition, a photograph was taken on day 4 of a mesoderm cell (adherent cell) differentiated from an induced pluripotent stem cell colony. In addition, a photograph was taken on day 12 of a hematopoietic stem cell (a floating cell existing as a single cell) differentiated from a feeder cell (a part where cells are attached and clumped together) and a mesoderm cell (adherent cell). In addition, a photograph was taken on days 26 to 33 of a natural killer cell differentiated from a hematopoietic stem cell (autologous supporting cells are excluded in the photograph). Finally, a photograph was taken on day 48 of a natural killer cell (a floating cell) derived from an induced pluripotent stem cell.

[0266] Furthermore, after changing the culture conditions, the results obtained were confirmed by observing the morphological changes of the differentiated cells at each differentiation stage for each condition (A-D) using an optical microscope (Figure 13). This time, pictures were taken under the microscope at each differentiation stage, from day 0 of differentiation into natural killer cells to the final day of differentiation.

[0267] Example 1.6. Confirmation of the generation of natural killer cells in the presence or absence of iPSC-derived feeder cells and confirmation of the properties of the generated natural killer cells During the methods of Examples 1.1 to 1.4 above, the generation of mesodermal cells and natural killer cells was confirmed depending on the presence or absence of iPSC-derived feeder cells (FIG. 4).

[0268] The figure above shows a photograph of an entire culture flask of mesodermal cells derived from an induced pluripotent stem cell colony. In this study, the size of the induced pluripotent stem cell colony at the beginning of differentiation was impossible to capture with a general camera, so the entire culture vessel could only be captured at the mesodermal cell differentiation stage, which had been differentiated for 3 to 4 days.

[0269] Furthermore, the middle figure shows a picture of the whole flask after differentiation into hematopoietic stem cells was completed (differentiation day 12) and after differentiation into natural killer cells was performed (differentiation day 25) in the group where only the autologous feeder cells were removed and in the group where the autologous feeder cells were maintained. Finally, the bottom figure shows a picture of the completion stage of autologous feeder cell differentiation.

[0270] Furthermore, hematopoietic stem cells and natural killer cells were photographed according to the presence or absence of feeder cells (Figure 5). Specifically, photographs were taken from day 0 of differentiation into natural killer cells to the day of terminal differentiation, up to day 45, and on day 12 of differentiation (the stage at which hematopoietic stem cell differentiation is completed), the group in which the autologous feeder cells were removed and the group in which the autologous feeder cells were maintained were photographed and compared.

[0271] Furthermore, the total amount of floating cells obtained and the expression rate of natural killer cell surface marker proteins (markers) at each differentiation stage in the group in which autologous feeder cells were maintained and in the group in which the autologous feeder cells were removed were confirmed by flow cytometry (Figures 6 and 7).

[0272] Example 1.7. Analysis of natural killer cell yield according to the number of iPSCs seeded To confirm the proliferation ability according to the number of seeded iPSCs, the number of iPSCs was varied in the culture medium and cultured according to the methods of Examples 1.1 to 1.4 above. The number of iPSCs at the time of seeding can be confirmed through the number of cultured iPSC colonies.

[0273] Specifically, the number of cells was measured using the following method. First, the medium in the T-150 flask was removed, then washed with 1 mL of DPBS. 10 ml of TryLE Express was added and reacted in a 37°C incubator for 15 minutes. The colonies attached to the scraper were collected and obtained in a 50 ml tube. This time, the colonies were collected after thoroughly checking that no colonies remained. After washing with 15 ml of complete medium to ensure that all cells remaining in the flask could be collected, the cells were collected in a tube. Step 4 was repeated once more to make a total of 40 ml.

[0274] Centrifugation was performed at 300g for 5 min. The supernatant was removed, the cells were released by tapping, and the cells were then resuspended in 1 mL of complete medium by pipetting. 9 mL of complete medium was added to bring the total volume to 10 mL. The cells were then counted.

[0275] Specifically, iPSCs were grown under the conditions described in Table 1 below to generate natural killer cells. [Table 1] In this case, Figure 8a shows the results obtained by analyzing the number of attached iPSC colonies under each of the above conditions. Specifically, induced pluripotent stem cell colonies were formed differentially under each condition, and then the entire culture vessel was photographed at the mesoderm cell stage (differentiation days 3-4). The number of iPSC colonies and the size of the colonies were measured. As a result, induced pluripotent stem cell colonies on differentiation day 0 could not be photographed at the corresponding magnification, and there was no difference in the number of colonies on differentiation days 0 and 4. However, it was confirmed that the size of the iPSC colonies on day 4 was larger. The measurement results in Figure 8a above were analyzed, and the numerical value representing the total number of cells / attached vessel area (cm) was calculated. 2 ) (Figures 8b-8c).

[0276] Furthermore, the morphological changes of the cells differentiated at each stage under each of conditions 1 to 6 were confirmed using an optical microscope (Figure 9).

[0277] Furthermore, the amount of differentiated natural killer cells (suspension cells, day 35) for each condition was confirmed (FIG. 10), and the yield of suspension cells, which are differentiated natural killer cells, was confirmed for each condition based on the amount of suspension cells produced under condition 2 (Table 2). Furthermore, the results were analyzed based on the results of condition 2 as the amount of suspension cells under each condition, as "initial differentiated pluripotent cell colonies / volume of culture medium" (Table 3) and "initial differentiated pluripotent cell colonies / differentiation area" (Table 4). [Table 2] [Table 3] [Table 4] To confirm the characteristics of iPSC-derived natural killer cells induced to differentiate under each of the above conditions 1 to 6, the expression rate of natural killer cell surface marker proteins (markers) was confirmed by flow cytometry, and the results are shown in FIG. 11. When differentiation was induced, the total number of natural killer cells (floating cells) obtained under the above conditions was confirmed. Compared to the above conditions 1 to 6, smaller iPSC colonies were formed, and then differentiation into mesodermal cells was induced. As a result, as shown in FIG. 12, FIG. 13, and Table 5, it was confirmed that differentiation into mesodermal cells is possible even for iPSC colonies with a size of 300 μm to 400 μm (FIGS. 12 to 14 and Table 5). [Table 5] Example 1.8. Reproducibility experiment on the generation of natural killer cells from induced pluripotent stem cell colonies Differentiation of induced pluripotent stem cells into natural killer cells was repeatedly performed under the same conditions as condition 2 above, and the experimental results were summarized (Figures 15 to 18).

[0278] Example 1.9. Generation of natural killer cells from induced pluripotent stem cell colonies following culture medium changes Natural killer cells were generated from induced pluripotent stem cells by the differentiation methods described above in Examples 1.1 to 1.4. This time, natural killer cells were generated in the same manner, except that Stempro34 medium was used (Figure 19).

[0279] When differentiation of iPSCs into natural killer cells was induced by the above-mentioned method, surface marker proteins (markers) of natural killer cells were identified in the cells obtained at each time point by flow cytometry (Figures 20 to 25).

[0280] Example 1.10. Mass Expansion of Natural Killer Cells Derived from Induced Pluripotent Stem Cells The natural killer cells obtained in the same manner as in Examples 1.1 to 1.4 above were expanded by a mass culture process. The mass culture process was carried out once every 2 to 3 days. Specifically, the mass culture process was carried out on days 0, 3, 5, 7, 10, 12, 14, 17, 19, and 21, and the cells were finally harvested on day 21.

[0281] The culture medium used in the large-scale culture process was prepared by adding additional components to the basal medium described in Table 6. Specifically, the basal medium prepared in step 1 consisted of DMEM, Ham's F12 nutrient mixture, human AB serum, penicillin-streptomycin, and L-glutamine. Then, cytokines and factors selected from β-mercaptoethanol, sodium selenite, ethanolamine, L-ascorbic acid, SCF, IL-7, IL-15, FLT-3 ligand, and IL-2 were added to the basal medium and used. Hereinafter, the "culture medium" was referred to as the "culture solution." [Table 6] In the large-scale culture process, first, on the day when differentiation was completed (day 0), the first irradiated feeder cell stimulation was performed on the differentiated natural killer cells. Specifically, natural killer cells and irradiated feeder cells were co-cultured in a 1:2 ratio in a 6-well plate containing 2 ml of culture medium. On the third day of culture, half of the culture medium, 1 ml, was replaced. This time, the cells in the replaced culture medium were collected and cultured again. On the fifth day of culture, half of the culture medium, 1 ml, was replaced, and then 3 ml of new culture medium was added to increase the volume to 4 ml. Furthermore, in the subsequent culture process, when all steps were performed until the 14th day of culture, half of the culture medium was replaced, the cells were collected, and the volume of the culture medium was increased.

[0282] When the culture vessel was replaced with a T-25 flask, half of the medium, 2 ml, was replaced and then 10 ml of fresh medium was added to bring the volume up to 12 ml. This time, the cells attached to the bottom of the 6-well plate were also collected using a 24 mm scraper (TPP, 99002), counted, and then transferred to a T-25 flask.

[0283] On the 10th day of culture, the culture vessel was changed from a T-25 flask to a T-75 flask. This time, half of the culture medium, 6 ml, was replaced, and then 24 ml of fresh culture medium was added to increase the volume of the culture medium to 30 ml. The cells attached to the bottom of the T-25 flask were also collected and transferred using a 30 mm scraper (TPP, 99003). On the 12th day of culture, the culture vessel was changed from a T-75 flask to a T-150 flask. This time, half of the culture medium, 15 ml, was replaced, and then 60 ml of fresh culture medium was added to increase the volume of the culture medium to 75 ml. Once the culture vessel was changed, the cells attached to the bottom of the T-75 flask were also collected and transferred using a 30 mm scraper (TPP, 99003) to a T-150 flask.

[0284] On day 14 of culture, the culture vessel was replaced with a 350 ml CO2-permeable bag. This time, half of the culture medium (38 ml) was replaced, and then 151 ml of fresh medium was added to increase the volume of the culture to 188 ml. In addition, the cells attached to the bottom of the T-150 flask were also collected using a 30 mm scraper (TPP, 99003) in the same manner as above, counted, and then transferred to a 350 ml CO2-permeable bag. This time, the above steps were performed using a 50 ml Luer Lock syringe (NORM-JECT, 48500003000).

[0285] On day 17 of culture, the volume of the culture medium was increased to 470 ml using a 50 ml Luer Lock syringe. From this point onwards, half of the medium was added continuously without replacement. On day 19 of culture, the culture vessel was replaced with a 1 L CO2-permeable bag and the volume of the medium was increased to 1175 ml.

[0286] On the 21st day of culture, the cells cultured in the 1 L CO2 permeable bag were finally harvested. In this specification, the natural killer cells induced to differentiate from the induced pluripotent stem cells were referred to as "EiNK cells".

[0287] II. Characterization of the generated natural killer cells Experimental Example 1. Verification of the expression of key markers of natural killer cells derived from induced pluripotent stem cells iPSC-derived natural killer cells produced by the methods of Examples 1.1 to 1.4 above were obtained by the method of Example 1.7 above, the total number of floating cells was confirmed, and then the expression of key markers for natural killer cells was confirmed by flow cytometry.

[0288] Specifically, NK cells were obtained by the methods of Examples 1.1-1.4 above, and then the cell pellet was resuspended in FACS buffer (Invitrogen, 00-4222-57). The total number of cells was counted by the method of Example 1.7, and then 1 × 10 5 The cells were placed in a FACS tube and suspended in 1 μl of FACS antibody presented in Table 7 per 100 μl of FACS buffer. The tube was wrapped in aluminum foil to block light, and staining was carried out at refrigerated temperature for 1 hour.

[0289] 1mL of FACS buffer was added to the stained cells, centrifuged at 300g for 5 minutes, and then the supernatant was removed. The pellet was resuspended again by adding 1mL of FACS buffer, and then centrifuged at 300g for 5 minutes twice. Finally, the cells were resuspended in 500μl of FACS buffer, and then the expression of cell surface markers was measured using a NovoCyte™ flow cytometer (Agilent, NovoCyte 3005) and analyzed using NovoExpress software (Agilent, Ver.1.5.0). [Table 7] Experimental Example 2. Evaluation of the cytotoxicity of natural killer cells derived from induced pluripotent stem cell colonies The cytotoxic function of natural killer cells derived from induced pluripotent stem cells prepared by the methods of Examples 1.1 to 1.4 above was evaluated using Abcam's cytotoxicity assay kit (Abcam, ab270780). Specifically, 1 × 10 5 The cells were suspended in 180 μl of measurement buffer (Abcam, ab270780) + 20 μl of CSFE solution (Abcam, ab270780), and then reacted at room temperature for 15 minutes to perform CSFE staining.

[0290] Centrifugation was performed at 1,000 rpm for 3 minutes, and then the supernatant was removed. The K562 cells that had completed CSFE staining and the induced pluripotent stem cell-derived natural killer cells (EiNK cells) prepared by the methods of Examples 1.1 to 1.4 above were mixed in 400 μl of RPMI1640 (Hyclone, SH30027.01) culture medium containing 10% FBS (Gibco, 10100147) at cell ratios of 1:10, 1:5, 2:1, and 1:1, respectively, and cultured in a cell incubator for 4 hours.

[0291] After the incubation was completed, 20 μl of 7-AAD (Abcam, ab270780) solution was mixed with the cell suspension and reacted at 4° C. for 10 minutes, and then the cytotoxicity level was confirmed using NovoCyte™ flow cytometry (Agilent, NovoCyte 3005) and analyzed using NovoExpress software (Agilent, Ver.1.5.0). The results are shown in FIG. 26.

[0292] Experimental Example 3. Characterization of short-term massively expanded natural killer cells Experimental Example 3.1. Morphological verification of generated natural killer cells Mass proliferation of iPSC-derived natural killer cells (EiNK cells) prepared by the methods of Examples 1.1 to 1.4 above was performed by the method of Example 1.10, and the morphological changes of the cultured natural killer cells were observed using an optical microscope. Specifically, the morphology of the natural killer cells was confirmed using an optical microscope (Olympus, Olympus-CKX53) during the culture time after the first, second, and third co-culture with feeder cells and during the 21-day mass proliferation process according to the process of mass proliferation of induced pluripotent stem cell-derived natural killer cells (FIG. 28).

[0293] Experimental Example 3.2. Confirmation of yield of natural killer cells derived from induced pluripotent stem cells through mass expansion process The yield of induced pluripotent stem cell-derived natural killer cells (EiNK cells) generated by the same methods as in Examples 1.1-1.4 above was analyzed using the following method during mass expansion culture by the method of Example 1.10.

[0294] Specifically, during the mass expansion process of induced pluripotent stem cell-derived natural killer cells and after completion of the mass culture, all cells suspended in the cell culture vessel were obtained together with natural killer cell culture medium and centrifuged at 300 g for 5 minutes using a centrifuge (Labogene, 1580R). The cell pellet obtained by centrifugation was resuspended in natural killer cell culture medium, and then the total number of cells in the suspension was counted using an Incyto c-chip (Incyto, DHC-N01-5), and the results are shown in Figure 29 and Table 8. [Table 8] Experimental Example 3.3. Verification of the expression of cell markers in expanded natural killer cells derived from induced pluripotent stem cells Induced pluripotent stem cell-derived natural killer cells (EiNK cells) produced by the same method as in Examples 1.1 to 1.4 above were obtained by the method in Example 1.7 above, and the total number of floating cells was confirmed, and the expression of key markers for natural killer cells was confirmed by flow cytometry.

[0295] Specifically, large amounts of cultured natural killer cells derived from induced pluripotent stem cells were obtained, and then the cell pellets were resuspended in FACS buffer (Invitrogen, 00-4222-57). 5 The resuspended cells were placed in a FACS tube, then 1 μl of the respective antibody that binds to the cell marker (activation marker protein) was added per 100 μl of staining buffer, the tube was wrapped in aluminum foil to block light, and then staining was carried out at refrigerated temperature for 1 hour.

[0296] 1mL of staining buffer was added to the cells that had completed staining, centrifuged at 300g for 5 minutes to remove the supernatant, and then resuspended by adding 1mL of staining buffer. The resuspension was centrifuged at 300g for 5 minutes. The above resuspension and centrifugation steps were repeated twice. Finally, the cells were resuspended in 500μl of staining buffer, and the expression of cell markers was measured using NovoCyte™ flow cytometry (Agilent, NovoCyte 3005), and the measured values ​​were analyzed using NovoExpress software (Agilent, Ver.1.5.0). This time, the antibodies used are shown in Tables 7 and 9. In addition, anti-IgG1 antibody (BD, 555751) and anti-IgG2a, κ antibody (BD, 559319) were additionally used. [Table 9] As a result, as shown in Figures 30a and 30b, the expression rate of NKG2D, one of the natural killer cell activation receptors, was about 90% or higher. NKG2D is an important receptor for recognizing heterologous tumor cells, and is also an essential receptor in combination therapy with anticancer drugs that increase the expression of NKG2D ligands in tumor cells. In addition, the expression rates of DNAM1 and NKp30 were confirmed to be about 90%. The expression rate of CD16, which is involved in antibody-dependent cell-mediated cytotoxicity (ADCC) of natural killer cells, was about 50%. This time, this receptor plays a role in mediating other forms of MHC unlimited killing.

[0297] Experimental Example 3.4. Confirmation of the natural killer cell characteristics of expanded natural killer cells derived from induced pluripotent stem cells To confirm the killing ability of induced pluripotent stem cell-derived natural killer cells (EiNK cells) prepared in the same manner as in Examples 1.1 to 1.4 above against tumor cells, EiNK cells were obtained on the 21st and 28th days of mass expansion by the method of Example 1.7. The obtained natural killer cells were counted and co-cultured with a blood cancer cell line (K562) stained with the fluorescent substance CFSE (Abcam, ab270780). This time, the fluorescently stained blood cancer cell line was prepared by the following method. First, CFSE was diluted 1:250, and K562 cells were treated with CFSE and then reacted at room temperature for 15 minutes.

[0298] 1mL of cell culture medium was added to the stained cells, centrifuged at 2,000 rpm for 3 minutes, and then the supernatant was removed. 2mL of cell culture medium was added to the cells again, reacted at 37℃ in a 5% CO2 incubator for 30 minutes, and then centrifuged at 2,000 rpm for 3 minutes to obtain stained K562 cells. The number of K562 cells and natural killer cells prepared by the above method were counted, seeded into each well according to the E to T ratio, and co-cultured for 4 hours.

[0299] After 4 hours of co-culture, all the cells in the co-culture were placed in a FACS tube and then centrifuged at 2,000 rpm for 3 minutes. After centrifugation, the cells were resuspended by adding 1 mL of saline, then centrifuged again at 2,000 rpm for 3 minutes and the supernatant was removed. The above resuspension and centrifugation were repeated twice. After that, the cells were suspended in 200 μl of staining buffer, treated with 7-AAD (Abcam, ab270780), and then reacted on ice for 10 minutes.

[0300] After the reaction was completed, the cells stained with CFSE and 7-AAD simultaneously were analyzed using a NovoCyte™ flow cytometer (Agilent, NovoCyte 3005). The measured values ​​were analyzed using NovoExpress software (Agilent, Ver. 1.5.0), and the results are shown in Figure 31 and Table 10. [Table 10] Experimental Example 3.5. Confirmation of the natural killer cell characteristics of expanded natural killer cells derived from induced pluripotent stem cells Induced pluripotent stem cell-derived natural killer cells (EiNK cells) cultured in large quantities for 26 days in the same manner as in Example 1.10 above were obtained in the same manner as in Experimental Example 3.4 above, the total number of floating cells was confirmed, and the expression of iPSC markers (marker proteins, Table 11) in the obtained cells was confirmed by flow cytometry.

[0301] For flow cytometry, specifically, natural killer cells prepared by the method of Example 1 were obtained, and then the cell pellet was resuspended in FACS buffer (Invitrogen, 00-4222-57). The total number of cells was counted, and then 1×10 5The cells were placed in a FACS tube. 1 μl of the antibodies shown in Table 5 per 100 μl of FACS buffer was added and suspended. The tube was wrapped in aluminum foil to block light, and then the cells were stained by reacting at refrigerated temperature for 1 hour. After the staining was completed, 1 mL of FACS buffer was added to the cells, and centrifugation was performed at 300 g for 5 minutes. After removing the supernatant, the cell pellet was resuspended in 1 mL of FACS buffer and centrifuged again at 300 g for 5 minutes to wash the cells. The washing process was repeated twice in total. Finally, the cells were resuspended by adding 500 μl of FACS buffer, and then the expression of iPSC markers on the cell surface was measured using a NovoCyte™ flow cytometer (Agilent, NovoCyte 3005), and the measured values ​​were analyzed using NovoExpress software (Agilent, Ver.1.5.0).

[0302] As a result, it was confirmed that the expression rate of induced pluripotent stem cell marker factors in mass-cultured natural killer cells derived from induced pluripotent stem cells was 1% or less, as shown in Figure 32. Based on the above results, it was confirmed that almost no induced pluripotent stem cells remained in the cells after differentiation induction. [Table 11] Experimental Example 4. Characterization of long-term, massively expanded natural killer cells derived from induced pluripotent stem cells Experimental Example 4.1. Confirmation of the yield of long-term mass-expanded natural killer cells derived from induced pluripotent stem cells The EiNK cells prepared by the methods of Examples 1.1 to 1.4 above were cultured for up to 63 days, and then the proliferation rate of the EiNK cells was confirmed. This time, the EiNK cell medium was used by adding β-mercaptoethanol (Gibco, 21985-023), sodium selenite (Sigma, S5261-100G), L-ascorbic acid (Sigma, A4544-100G), and IL-15 (R&D systems, 247-ILB-500 / CF) to RPMI1640 (Cytiva, SH30027.01) containing human AB serum (Gemcell, 100-512) and penicillin-streptomycin (Gibco, 15140-122) (Table 12). [Table 12] EiNK cells were stimulated with irradiated feeder cells every 7 days and cultured for up to 63 days. As a result, as shown in Figure 33, EiNK cells cultured for up to 63 days showed a proliferation rate of 6.5 x 10 of the initially cultured cells. 10 The results were confirmed as logarithmic values, and it was confirmed that the cells were proliferated while maintaining a constant period-specific proliferation fold.

[0303] Experimental Example 4.2. Confirmation of cell-killing ability of long-term mass-expanded iPSC-derived natural killer cells The phenotype of EiNK cells prepared in the same manner as in Experimental Example 4.1 above, as well as the natural killer cell phenotype and killing ability against K562 cells, were confirmed. The phenotype was confirmed in the same manner as in Experimental Example 3.3 and Experimental Example 3.5, and the killing ability was confirmed in the same manner as in Experimental Example 3.4.

[0304] As a result, as shown in Figure 34a, the expression rate of CD56+CD45, which is a natural killer cell phenotype, was confirmed to be 99%, and the expression rate of CD56-CD3+, which is a T cell phenotype, was confirmed to be 0.06%, indicating that there were almost no T cells in the EiNK cells cultured for 63 days. Furthermore, the expression rate of induced pluripotent stem cell phenotype (TRA-1-60+, SSEA-4+) in the EiNK cells cultured for 63 days was confirmed to be 1% or less, indicating that there were almost no induced pluripotent stem cells remaining.

[0305] Furthermore, as shown in Figure 34b, the cell-killing ability of EiNK cells cultured for 20, 56, and 63 days against cancer cells was examined, and a high level of cancer cell-killing ability was observed regardless of the culture period. Based on the above results, it was confirmed that the EiNK cells of the present invention maintain high cytotoxicity even after long-term culture.

[0306] Experimental Example 5. Confirmation of anti-cancer activity of iPSC-derived natural killer cells: in vivo The anti-cancer activity of EiNK cells (cultured for 21 days) cultured in large quantities by the method of Example 1.10 above was confirmed at the animal level. This time, 6-week-old female NSG mice were used.

[0307] Specifically, luciferase was overexpressed in OVCAR3 cells, an ovarian cancer cell line, and 2 × 10 6 Cells / 100 μl (PBS, vehicle) were administered intraperitoneally to each mouse. EiNK cells used as test substances were thawed 2 days before administration, stabilized in T-75 flasks for 2 days, and then 1 × 10 7 Cells / 100 μl (PBS, vehicle) were intraperitoneally administered to each mouse. In this study, RPMI medium containing 5%-15% human AB serum, 1% L-glutamine, 50 IU / ml IL-2, and 10 ng / ml IL-15 was used as the culture medium for the thawed EiNK cells. The vehicle-treated group was used as the control.

[0308] The test substance (EiNK) was administered 12 times in total on days 2 (day 0), 4 (day 2), 6 (day 4), 9 (day 7), 11 (day 9), 13 (day 11), 16 (day 14), 18 (day 16), 20 (day 18), 23 (day 21), 25 (day 23), and 27 (day 25) after tumor cell administration. At the same time, IL-2 (10,000 IU / mouse) and IL-15 (0.5 μg / mouse) were administered intraperitoneally at intervals of 2 to 3 days for 28 days. After administration of the test substance, the body weight of the mice was measured once every 7 days from day 0, for a total of 3 times. Furthermore, luciferin was administered into the right abdominal cavity of the mice on days 0, 3, 7, 10, 14, 17, 21, 24, and 28, and tumor cells present in the mice were identified after 10 minutes of reaction using an IVIS imaging device.

[0309] As a result, as shown in Figures 40a to 40e, it was confirmed that in the control (solvent), luciferase activity rapidly increased after the 17th day. This result suggests that the OVCAR3 cells administered into the peritoneal cavity gradually proliferated in vivo over time. On the other hand, in the test substance treatment group, tumor cells continued to decrease from the day after the administration of the test substance (day 0), and after the 3rd day, almost no tumor cells were found proliferating in the peritoneal cavity. Based on the above results, it was confirmed that EiNK cells have anti-cancer activity at the animal level.

[0310] III. Comparison of iPSC-derived natural killer cells and blood-derived natural killer cells Experimental Example 6. Comparison of cell surface marker expression between mass-expanded iPSC-derived natural killer cells and blood-derived natural killer cells The numbers of EiNK cells cultured in large quantities by the method of Example 1.10 above (cultured for 21 days); and natural killer cells isolated from blood and then cultured by the method of Example 1.10 (PBNK, cultured for 22 days) were counted, and the expression of natural killer cell surface marker proteins (markers) was confirmed by flow cytometry.

[0311] This time, the expression of cell surface markers was analyzed using the BD Lyoplate human cell surface screening panel (BD, 560747). A panel of 242 antibodies against human cell surface markers was used to label EiNK and PBNK cells.

[0312] Specifically, EiNK cells were harvested and counted on day 21 of bulk culture, and PBNK cells were harvested and counted on day 22 of culture. Cells were then cultured at 2.5 × 10 6 The cells were resuspended in staining buffer (FACS buffer) at a concentration of 2.5 × 10 cells / ml. 5 Cells were seeded into each well of a 96-well plate at a density of 100 cells / well.

[0313] Each seeded cell was labeled with 242 primary antibody for 30 minutes, then the cells were washed with staining buffer and centrifuged (300g for 4 minutes) to remove the supernatant. The primary antibody was washed, and then Alexa647-labeled secondary antibody (mouse or rat) was added to each primary antibody and reacted for 30 minutes. After the secondary antibody reaction, the cells were washed with staining buffer and centrifuged at 300g for 4 minutes to remove the supernatant. The cells that had completed the staining were measured using a NovoCyte™ flow cytometer (Agilent, NovoCyte 3005), and the measured values ​​were analyzed using NovoExpress software (Agilent, Ver.1.5.0).

[0314] The results of cell surface markers showing differences between EiNK cells expanded in large numbers for 21 days and PBNK cells cultured for 22 days are shown in Figures 35a and 35b and Tables 13 and 14. [Table 13] [Table 14] As a result, it was confirmed that 26 of the 242 surface markers were increased in expression in EiNK cells compared to PBNK cells. The markers that increased more than 20-fold compared to PBNK cells were CD25, CD9, CDw327, and CD30, and the markers that increased more than 10-fold compared to PBNK cells were CD80 and CD114. Furthermore, the markers that increased more than 5-fold compared to PBNK cells were CD124, MIC A_B, CD210, CD87, CD123, CD83, and CD146, and the markers that increased more than 1.3-fold compared to PBNK cells were CD106, CD196, CD33, CD150, CD49e, CD108, CD49f, CD164, CD49c, CD66(a,c,d,e), CD49a, CDw93, and CD71. In contrast, seven markers were expressed exclusively in blood-borne natural killer cells.

[0315] Experimental Example 7. Comparison of gene expression between induced pluripotent stem cell-derived natural killer cells and blood-derived natural killer cells EiNK cells cultured in large quantities by the method of Example 1.10 above (cultured for 28 days); and natural killer cells isolated from blood and then cultured by the method of Example 1.10 (PBNK, cultured for 22 days), RNA was extracted from the natural killer cells and subjected to transcriptome sequencing and data analysis using QuantSeq 3'mRNA-Seq. Whole genome RNA sequencing analysis was outsourced to Ebiogen (Korea). Sequencing results were analyzed for gene expression changes using ExDEGA (an Excel-based differentially expressed genes tool).

[0316] As a result, as shown in FIG. 36, it was confirmed that the expression of factors involved in tumor cell killing and immune activation was increased in EiNK cells compared to PBNK cells.

[0317] Experimental Example 8. Comparative analysis of chemokines and cytokines secreted in the culture medium of iPSC-derived natural killer cells and blood-derived natural killer cells EiNK cells cultured in bulk by the method of Example 1.10 above (cultured for 28 days); and natural killer cells isolated from blood and then cultured by the method of Example 1.10 (PBNK, cultured for 22 days) were analyzed for cytokines in the culture medium. Cytokine analysis was performed using the Proteome Filter Human XL Cytokine Array Kit (R&D Systems, Catalog No. ARY022B) according to the manufacturer's protocol. Results of the cytokine array kit measurements were analyzed using GelQuantNet software (BiochemLabSolutions) for comparative dot intensity against reference control dots. The cytokines, chemokines, and growth factors used in the cytokine array are listed in Table 15.

[0318] Specifically, the qualitative analysis of 102 cytokines, chemokines, or cell growth factors was carried out according to the following steps. After all the reagents and culture medium samples of the provided product were prepared at room temperature, Array Buffer 6 and membrane were added to each well of a pre-designed 4-well plate and reacted on a rocking shaker for 1 hour at room temperature. Then, the solution was discarded and the culture medium sample was added together with Array Buffer 6 to a final volume of 1.5 ml (the culture medium sample was used diluted to 1 / 10 of the original concentration). After reacting for 12 hours or longer while maintaining at 2-8°C on a rocking shaker, the mixture was washed with washing solution three times for 10 minutes each. The detection antibody cocktail (60 μl) was added to the mixture (6 ml) of the provided Array Buffer 6 (4 ml) and Array Buffer 4 (2 ml), and each well was treated with 1.5 ml and reacted at room temperature for 1 hour. Then, the wells were washed with washing solution three times for 10 minutes each, streptavidin-HRP was added to each well and reacted for 30 minutes, then the wells were washed with washing solution three times for 10 minutes each, Chemi reagent mixture was added and reacted for 1 minute, and the luminescence level was confirmed (Chemi-doc photo).

[0319] As a result, as shown in Figures 37a and 37b, it was confirmed that the concentrations of chemokines and cytokines released by activated natural killer cells in EiNK cell culture medium were higher than those of PBNK cells. [Table 15] Experimental Example 9. Comparative analysis of the expression of inhibitory receptors and cell surface markers in iPSC-derived natural killer cells and blood-derived natural killer cells Expression of natural killer cell inhibitory receptors and cell surface markers was confirmed by flow cytometry in EiNK cells cultured in large quantities by the method of Example 1.10 above (cultured for 21 days); and natural killer cells isolated from blood and then cultured by the method of Example 1.10 (PBNK, cultured for 21 days). The above experiment was repeated a total of four times.

[0320] Cells were obtained and used in the same manner as in Experimental Example 3.4, and FACS analysis was performed according to the method of Experimental Example 3.5, but this time the antibodies used in the analysis are listed in Tables 7, 11, and 16. [Table 16] As a result, as shown in Figure 38, NKG2A / CD94PBNK, a marker protein expressed in mature natural killer cells, showed similar expression rates in PBNK cells and EiNK cells. On the other hand, TIM-3 and TIGIT are known to have the function of suppressing immune activity as inhibitory receptors, but it was confirmed that their expression rates were reduced in EiNK cells compared to PBNK cells. Based on the above results, it is considered that the immune activation ability of EiNK cells is superior to that of PBNK cells.

[0321] Experimental Example 10. Comparison of the tumor cell killing ability of iPSC-derived natural killer cells and blood-derived natural killer cells The tumor cell killing ability of natural killer cells was confirmed for EiNK cells (cultured for 21 or 28 days) cultured in large quantities by the method of Example 1.10 above; and natural killer cells (PBNK, cultured for 16 days) isolated from blood and then cultured by the method of Example 1.10. Each cell was obtained by the method of Example 3.4, and the tumor cell killing ability was confirmed by the method of Example 3.4. The above experiment to confirm the killing ability was repeated a total of seven times.

[0322] As a result, it was confirmed that the tumor cell killing ability of EiNK cells and PBNK cells was at a similar level, as shown in Figure 39. However, while PBNK cells showed differences in cell killing ability depending on the blood donor, EiNK cells showed a similar level of tumor cell killing ability depending on the batch culture.

[0323] IV. Comparison of the method for differentiating induced pluripotent stem cell-derived natural killer cells of the present invention with conventional methods for differentiating induced pluripotent stem cell-derived natural killer cells Experimental Example 11. Comparison of cell yields and proliferation rates following CD34+ sorting FIG. 42a shows the results obtained by examining the changes in cell yield, cell surface marker expression, and cell proliferation rate due to the sorting step of CD34-positive cells, a differentiation marker of hematopoietic stem cells, during a conventional method for differentiating induced pluripotent stem cell-derived natural killer cells.

[0324] Specifically, induction of natural killer cell differentiation from conventional iPSCs was carried out as follows.

[0325] For differentiation of induced pluripotent stem cells (iPSCs), iPSCs were seeded in Aggrewell plates (aggrewell 400, STEMCELL, 34425), and then embryoid body (EB) formation was induced for 5 days by replacing EB-A medium (STEMdiff™ NK Cell Kit, STEMCELL, 100-0170) every day. This time, only 50% of the existing medium was removed, and new medium was added in the amount removed. To collect only EBs larger than a certain size, the medium containing EBs was passed through a filter (reversible 37 μm, STEMCELL, 27215) to remove single cells or small-sized EBs. After collecting the filtered EBs through a strainer by washing the EBs with medium, the EBs were seeded in a non-tissue culture 6-well plate (STEMCELL, 38040) and cultured for 7 days by replacing the medium using EB-B medium (STEMdiff™ NK Cell Kit, STEMCELL, 100-0170) once every 2-3 days. This time, the medium replacement method was the same as above.

[0326] EBs cultured as described above were collected and reacted with collagenase (collagenase type IV, STEMCELL, 07909) for 20 min and TrypLE Select (Gibco, A12563011) for 20 min (total reaction with degradative enzymes for 40 min) to dissociate EBs into single cells. The isolated cells were selected for CD34-expressing cells using Easysep human CD34 positive selection kit II (STEMCELL, 17856). This time, sorting was performed by collecting floating cells, mixing the floating cells with anti-CD34 bound to magnetic bead antibody (CD34 Microbead Kit, Miltenyi Biotec, 130-046-702), loading it onto an LC column (Miltenyi Biotec, 130-042-401) attached to a MACS sorter, washing the column several times with DPBS (Cytiva, SH3002802), and collecting the cells remaining on the column.

[0327] The isolated cells were seeded in non-tissue culture 24-well plates (STEMCELL, 38042), and the medium was replaced once every 3-4 days. The medium replacement method was the same as above. From day 12 to day 19 after differentiation induction, lymphocyte differentiation medium (STEMdiff™ NK Cell Kit, STEMCELL, 100-0170) was used, and from day 19, lymphocyte progenitor cell proliferation medium (STEMdiff™ NK Cell Kit, STEMCELL, 100-0170) was used for culture. On day 26 of differentiation induction, all cells were collected, the number of cells was measured, and then the cells were re-seeded and cultured in non-tissue culture 24-well plates (STEMCELL, 38042). The medium was replaced once every 3-4 days as above. On day 41 of differentiation induction, all cells were collected, and flow cytometry analysis and cell counting were performed to confirm the results of natural killer cell differentiation.

[0328] As described above, when differentiation of induced pluripotent stem cells into natural killer cells was induced by the conventional method, mesodermal cells were induced to differentiate into hematopoietic stem cells, and then only CD34 positive cells (hematopoietic stem cells) were obtained and induced to differentiate into NK cells. When CD34 positive cells were selected and used in this method, the cell yield was significantly decreased, cell death was increased, and the expression rate of natural killer cell marker protein (CD56) and activation marker protein (NKp30) in the selected CD34 positive cells was decreased (Figures 42a and 42b). Furthermore, it was confirmed that the cell proliferation rate was also significantly decreased (Figure 42c).

[0329] On the other hand, the EiNK production method according to the present invention can induce differentiation into NK cells without CD34 sorting because the expression rate of CD34, a hematopoietic stem cell differentiation marker at the intermediate stage of NK differentiation, is high (>95%), thereby increasing cell yield and activity (Figure 42d).

[0330] Experimental Example 12. Comparison of cell viability following tertiary and secondary culture of iPSCs Figures 43 and 44 show the results obtained by ascertaining cell viability when the cells are obtained by culturing induced pluripotent stem cells using the third culture method.

[0331] Generally, the induced pluripotent stem cells were cultured using the third culture method and differentiation was induced using the method described in Experimental Example 11.

[0332] As shown in Figures 43 and 44, it was confirmed that when iPSCs were subjected to the third culture using Aggrewell, the size of the embryoid bodies was larger than that of the second culture. Furthermore, it was confirmed that there was a difference in the size of the embryoid bodies depending on the batch, and that the cell viability was reduced when single cells were obtained by treatment with degradative enzymes. Meanwhile, the method for differentiating EiNK cells according to the present invention is considered to be simpler and more efficient than the third culture, because the iPSCs were induced to differentiate by undergoing the second culture.

[0333] Experimental Example 13. Comparison of differentiation induction periods for natural killer cells derived from induced pluripotent stem cells Figures 45 and 46 show the results of comparing the time period for generating natural killer cells by a conventional EiNK differentiation method (general method) and the differentiation method according to the present invention (method of the present invention), and confirming the phenotype of EiNK generated according to the present invention. Conventional EiNK differentiation was performed in the same manner as described in Experimental Example 11.

[0334] The conventional EiNK differentiation method required a total differentiation induction period of 48 days, while the EiNK differentiation method according to the present invention required a differentiation induction period of 26 days. As a result of confirming natural killer cell marker proteins (CD56, CD45, NKp44) and activation proteins (CD56, NKp30) in EiNK cells induced to differentiate for 26 days, it was confirmed that the expression rate of NK marker proteins and activation proteins was 66% or higher. On the other hand, the expression rate of iPSC marker proteins (TRA-1-60, SSEA4) was found to be 0%, indicating that the EiNK cell differentiation method according to the present invention is an efficient method that can produce natural killer cells in a short period of time compared to conventional methods.

Claims

1. A natural killer cell expressing any one protein selected from the group consisting of CD25, CD9, CD80, and combinations thereof.

2. The natural killer cell of claim 1, wherein the natural killer cell further expresses any one protein selected from the group consisting of CD1d, CD30, CD87, CDw327, CD33, CD49a, CD49c, CD49e, CD49f, CD66a, CD66c, CD66d, CD66e, CD71, CD83, CDw93, CD106, CD108, CD114, CD123, CD124, CD146, CD150, CD164, CD196, CD210, and combinations thereof.

3. The natural killer cell of claim 1, wherein the natural killer cell expresses any one of the natural killer cell marker proteins selected from the group consisting of CD56, CD45, CD16, NKp30, NKp44, NKG2D, DNAM1, and combinations thereof.

4. The natural killer cell of claim 1, characterized in that the natural killer cell has increased expression of the protein of claim 1 or 2 compared to natural killer cells isolated from blood.

5. The natural killer cell of claim 4, wherein expression of the protein is increased by at least two-fold compared to natural killer cells isolated from blood.

6. The natural killer cell of claim 1, wherein the natural killer cell is characterized by an increased expression of any one of the genes selected from the group consisting of GZMB, CD226, RASGRP1, CD107a, CCR9, CD40LG, CD300c, and combinations thereof, compared to natural killer cells isolated from blood.

7. The natural killer cell of claim 1, wherein the natural killer cell is characterized by increased secretion of any one protein selected from the group consisting of FLT3L, MIP1 alpha / beta, PF4, IGFBP2, IGFBP3, MMP9, and combinations thereof, compared to natural killer cells isolated from blood.

8. The natural killer cell of claim 7, wherein secretion of the protein is increased by at least two-fold compared to natural killer cells isolated from blood.

9. The natural killer cell of claim 1, wherein the natural killer cell does not further express any one protein selected from the group consisting of CD205, CD158b, CD243, γδTCR, NKB1, CD158a, CDw328, TIM3, TIGIT, and combinations thereof, or expresses the same at a lower rate compared to natural killer cells isolated from blood.

10. The natural killer cell of claim 9, wherein expression of the protein is reduced by more than half compared to natural killer cells isolated from blood.

11. The natural killer cell of claim 1 , wherein the natural killer cell does not express CD3, a marker protein for T cells.

12. The natural killer cell of claim 1 , wherein the natural killer cell is differentiated from an induced pluripotent stem cell (iPSC).

13. A method for producing natural killer cells, comprising: 1 cm ２ seeding induced pluripotent stem cells (iPSCs) as iPSCs or iPSC spheroids into a cell culture vessel so that 0.5 to 20 colonies are formed per area of ​​the cell culture vessel, and performing adherent culture to generate iPSC colonies; Differentiating the iPSC colonies into mesodermal cells to generate mesodermal cells; Differentiating the mesodermal cells into hematopoietic stem cells to generate hematopoietic stem cells; and A step of differentiating hematopoietic stem cells into natural killer cells to produce natural killer cells. The natural killer cell of claim 12, produced by a method comprising:

14. The seeded iPSCs were cultured at 1 cm ２ The natural killer cells of claim 13, seeded at 1-8 iPSCs per cell culture vessel area.

15. The seeded spheroids were 1 cm ２ The natural killer cells of claim 13, seeded at 1 to 8 spheroids per cell culture vessel area.

16. The total number of stem cells on the day of differentiation was 1 cm ２ 1 x 10 per cell culture vessel area ３ ~1×10 ６ The natural killer cell of claim 13 .

17. The natural killer cell according to claim 13 , wherein the cells attached to the culture vessel are not removed during the step of differentiating the mesodermal cells into hematopoietic stem cells.

18. The natural killer cell according to claim 13, wherein the cells differentiated from mesodermal cells into hematopoietic stem cells have a CD34 protein expression rate of 85% or more without a CD34-positive cell sorting step.

19. The natural killer cell of claim 13, wherein the natural killer cell is produced by further performing mass culture expansion on natural killer cells induced to differentiate.

20. The natural killer cell of claim 19, wherein the large-scale culture expansion comprises culturing the natural killer cell induced to differentiate in a medium containing SCF (stem cell factor), FLT3L (FMS-like tyrosine kinase 3 ligand), IL-7, IL-15, IL-2, or a combination thereof.

21. 20. The natural killer cell of claim 19, wherein the large-scale culture expansion comprises culturing the natural killer cells induced to differentiate in a medium further comprising β-mercaptoethanol, sodium selenite, ethanolamine, L-ascorbic acid, or a combination thereof.

22. The natural killer cells of claim 19, wherein the mass culture expansion of the natural killer cells induced to differentiate is carried out for 1 day to 9 weeks.

23. A cell therapy comprising the natural killer cells according to any one of claims 1 to 22 as an active ingredient.

24. A pharmaceutical composition for preventing and treating diseases, comprising the natural killer cells according to any one of claims 1 to 22 as an active ingredient.

25. The pharmaceutical composition for preventing and treating a disease according to claim 24, wherein the disease is any one selected from the group consisting of an infectious disease, a cancer, a neurological disease, and an autoimmune disease.

26. 26. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein the infectious disease is any one selected from the group consisting of hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, and influenza.

27. 26. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein the cancer is any one selected from the group consisting of liver cancer, lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colorectal cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

28. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein the neurological disease is a neurological disease caused by nerve damage or abnormal nerves, or a degenerative brain disease.

29. The pharmaceutical composition for preventing and treating a disease according to claim 28, wherein the nerve damage is any one selected from the group consisting of transient conduction disorder, axotomy, or neurotomi.

30. The pharmaceutical composition for preventing and treating a disease according to claim 28, wherein the neurological disease caused by nerve damage or abnormal nerves is a neurological disease caused by nerve damage or abnormal nerves in the central nervous system, or a neurological disease caused by nerve damage or abnormal nerves in the peripheral nervous system.

31. 31. The pharmaceutical composition for preventing and treating a disease according to claim 30, wherein the neurological disease caused by nerve damage or abnormal nerves in the central nervous system is any one selected from the group consisting of organic diseases and functional disorders in the central nervous system, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Lewy body disease, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, spinocerebellar degeneration, cerebellar atrophy, post-traumatic stress disorder, amnesia, vascular dementia, and cerebral infarction.

32. 31. The pharmaceutical composition for preventing and treating a disease according to claim 30, wherein the neurological disease caused by nerve damage or abnormal nerves in the peripheral nervous system is any one selected from the group consisting of peripheral neuropathy, diabetic neuropathy, peripheral neuropathic pain, peripheral neuropathy caused by anti-cancer chemotherapy, complex regional pain syndrome, optic neuropathy, mononeuropathy, mononeuritis multiplex (mononeuropathy multiplex), polyneuropathy, Guillain-Barre syndrome (acute inflammatory demyelinating polyneuropathy), chronic inflammatory demyelinating polyneuropathy, hereditary neuropathy, plexopathy, glaucoma, macular degeneration, amyotrophic lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, poliomyelitis, post-polio syndrome, stiff-person syndrome, Isaacs syndrome, myasthenia gravis, neonatal myasthenia gravis, botulism, Eaton-Lambert syndrome, thoracic outlet syndrome, Charcot-Marie-Tooth disease, and spinal muscular atrophy.

33. 26. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein the neurological disease is a degenerative brain disease.

34. 34. The pharmaceutical composition for preventing and treating a disease according to claim 33, wherein the degenerative brain disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down's syndrome, amyloid stroke, systemic amyloid disease, Dutch-type amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedreich's ataxia, Machado-Joseph disease, Lewy body disease, dystonia, progressive supranuclear palsy, and frontotemporal dementia.

35. 26. The pharmaceutical composition for preventing and treating a disease according to claim 25, wherein the autoimmune disease is any one selected from the group consisting of Crohn's disease, erythema, atopy, rheumatoid arthritis, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, lupus, chronic fatigue syndrome, fibromyalgia, hypothyroidism and hyperthyroidism, scleroderma, Behcet's disease, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, Meniere's disease, Guillain-Barre syndrome, Sjogren's syndrome, vitiligo, endometriosis, psoriasis, vitiligo, systemic sclerosis, asthma, and ulcerative colitis.

36. 1. A method for producing natural killer cells, comprising: 1 cm ２ seeding induced pluripotent stem cells (iPSCs) as iPSCs or iPSC spheroids into a cell culture vessel so that 0.5 to 20 colonies are formed per area of ​​the cell culture vessel, and performing adherent culture to generate iPSC colonies; Differentiating the iPSC colonies into mesodermal cells to generate mesodermal cells; Differentiating the mesodermal cells into hematopoietic stem cells to generate hematopoietic stem cells; and A step of differentiating hematopoietic stem cells into natural killer cells to produce natural killer cells. The method includes:

37. 37. A method for producing the natural killer cells of claim 36, further comprising mass culture expansion of the natural killer cells induced to differentiate.

38. The total number of stem cells on the day of differentiation was 1 cm ２ 1 x 10 per cell culture vessel area ３ ~1×10 ６ A method for producing the natural killer cell of claim 36, which is a cell.

39. The method for producing natural killer cells according to claim 36, wherein the cells attached to the culture vessel are not removed during the step of differentiating the mesodermal cells into hematopoietic stem cells.

40. Use of the natural killer cells described in claim 1 for the prevention or treatment of a disease.

41. The use for prevention or treatment according to claim 39, wherein the disease is any one selected from the group consisting of infectious diseases, cancer, neurological diseases, and autoimmune diseases.

42. A method for preventing or treating a disease comprising administering the natural killer cells of claim 1.

43. 43. The method for preventing or treating a disease according to claim 42, wherein the disease is any one selected from the group consisting of an infectious disease, a cancer, a neurological disease, and an autoimmune disease.

44. 1 cm ２ 2. A method for culturing stem cells, comprising the step of seeding stem cells as iPSCs or iPSC spheroids into a cell culture vessel such that 0.5-20 colonies are formed per area of ​​the cell culture vessel.

45. A method for producing natural killer cells, comprising the step of differentiating hematopoietic stem cells into natural killer cells in the presence of feeder cells.

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