Highly efficient method for proliferating induced-NK cells (INKS)

Abstract

The present invention relates to a highly efficient method for proliferating induced-NK cells (iNKs). According to the present invention, it has been identified that iNK proliferation increases when feeder cells, into which dual plasmids containing mb4-1BBL and mbIL-21 genes are introduced, are co-cultured at a specific ratio in a medium for expanding natural killer cells containing cytokines such as interleukin-18 after differentiation from human induced pluripotent stem cells into natural killer cells. Therefore, through the method for expanding natural killer cells, according to the present invention, natural killer cells differentiated from human induced pluripotent stem cells are mass-proliferated, and are expected to be easily used in the development of various therapeutic agents for immune diseases and cancer.

Description

High-efficiency induced natural killer (INK) cell proliferation method

[0001] The present invention relates to a method for highly efficient induced natural killer (iNK) cell proliferation.

[0002] This application claims priority to Korean Patent Application No. 10-2024-0026804, filed February 23, 2024, and Korean Patent Application No. 10-2025-0022491, filed February 20, 2025, the entire disclosures of which are incorporated herein by reference.

[0003]

[0004] Natural killer cells (NK cells), which make up the immune system, account for approximately 10% of blood cells and have various functions, but they are particularly capable of killing cancer cells or cells infected with pathogens that have invaded from outside, thus eliminating abnormal cells that have become tumorigenic or are progressing into tumorigenicity. NK cells play a crucial role in the early stages of viral infection or tumorigenesis, before activated cytotoxic T lymphocytes are produced in large numbers. Histologically, NK cells are large granular lymphocytes. Intracellular granules contain molecules with pre-formed, potent biological functions that are secreted when NK cells come into contact with target cells. Some of these molecules form pores in the target cell membrane, lysing the cell or invading the target cell, increasing nuclear DNA fragmentation and inducing apoptosis or programmed cell death.

[0005] Natural killer (NK) cells are known to have the ability to nonspecifically kill cancer cells. This killing ability is being utilized in the treatment of solid tumors using lymphokine-activated killer cells (LAK) and tumor-infiltrating lymphocytes (TILs), and in immunotherapy using donor lymphocyte infusions to prevent rejection during bone marrow or organ transplantation, a novel cell therapy approach.

[0006] In addition, defects in the differentiation and activity of NK cells have been reported to be associated with various cancers, including breast cancer, melanoma, and lung cancer, and NK cell therapy is emerging to treat these diseases.

[0007] Effective use of NK cells as anticancer immunotherapy requires securing a large number of NK cells. However, NK cells account for 10-15% of lymphocytes in the blood, and cancer patients often have reduced numbers, differentiation, and function, making securing sufficient numbers of NK cells challenging. Therefore, mass production of NK cells through proliferation or differentiation is required for their application as NK cell therapy.

[0008] Meanwhile, induced pluripotent stem cells (iPSCs) are pluripotent cells derived from differentiated somatic cells, induced to the early embryonic stage through a process of dedifferentiation. Because iPSCs can differentiate into all cell types, they are actively being researched as cell therapies for intractable diseases without the risk of immune rejection using patient-derived cells.

[0009] Accordingly, the inventors of the present invention attempted to differentiate natural killer cells from stem cells such as induced pluripotent stem cells and co-culture them with genetically engineered feeder cells to mass-produce natural killer cells.

[0010]

[0011] The present inventors have developed a method for mass-producing induced natural killer cells (induced-NK cells, iNK) by producing a dual plasmid containing mb4-1BBL and mbIL-21, co-culturing feeder cells containing the dual plasmid after differentiation from human induced pluripotent stem cells into natural killer cells, and have completed the present invention based on this.

[0012] Accordingly, the purpose of the present invention is to provide a dual plasmid vector comprising a membrane bound human 4-1BB ligand (mb4-1BBL) and a membrane bound human interleukin-21 (mbIL-21) gene.

[0013] Another object of the present invention is to provide a feeder cell transfected with the dual plasmid vector.

[0014] Another object of the present invention is to provide a method for differentiating stem cells into natural killer cells, comprising a step of culturing isolated stem cells in a differentiation medium.

[0015] Another object of the present invention is to provide a step of culturing isolated stem cells in a differentiation medium to differentiate them into natural killer cells; and

[0016] A method for expanding natural killer cells is provided, comprising a step of co-culturing the natural killer cells with feeder cells of the present invention in a medium for expanding natural killer cells.

[0017] Another object of the present invention is to provide a composition for expanding natural killer cells, which comprises the feeder cells of the present invention as an effective ingredient.

[0018] Another object of the present invention is to provide a kit for natural killer cell expansion, which comprises the feeder cells of the present invention, a differentiation medium, and a natural killer cell expansion medium as active ingredients.

[0019]

[0020] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0021]

[0022] To achieve the above-described purpose, the present invention provides a dual plasmid vector comprising a membrane bound human 4-1BB ligand (mb4-1BBL) and a membrane bound human interleukin-21 (mbIL-21) gene.

[0023] As one embodiment of the present invention, the mb4-1BBL gene may include a base sequence of SEQ ID NO: 1, and the mbIL-21 gene may include a base sequence of SEQ ID NO: 2, but is not limited thereto.

[0024] As another embodiment of the present invention, the vector comprises mEF1α (Mouse elongation factor 1α), Neo r (neomycin resistance gene), and rEF1α (Rat elongation factor 1α), but is not limited thereto.

[0025] In addition, the present invention provides a feeder cell transfected with the dual plasmid vector.

[0026] In one embodiment of the present invention, the feeder cell may be, but is not limited to, K562 cells.

[0027] As another embodiment of the present invention, the feeder cells may include, but are not limited to, cells in which human leukocyte antigen (HLA) expression is inhibited.

[0028] In addition, the present invention provides a method for differentiating stem cells into natural killer cells, comprising a step of culturing isolated stem cells in a differentiation medium.

[0029] In addition, the present invention comprises a step of culturing separated stem cells in a differentiation medium to differentiate them into natural killer cells; and

[0030] A method for expanding natural killer cells is provided, comprising a step of co-culturing the natural killer cells with the feeder cells in a medium for expanding natural killer cells.

[0031] As one embodiment of the present invention, the stem cells may be human induced pluripotent stem cells (iPSCs), but are not limited thereto.

[0032] As another embodiment of the present invention, the human induced pluripotent stem cell may be, but is not limited to, a non-genetically modified human induced pluripotent stem cell or a genetically modified human induced pluripotent stem cell.

[0033] As another embodiment of the present invention, the genetically engineered human induced pluripotent stem cell may be a human induced pluripotent stem cell into which a chimeric antigen receptor (CAR) gene has been introduced, but is not limited thereto.

[0034] As another embodiment of the present invention, the differentiation medium may include, but is not limited to, one or more media selected from the group consisting of Essential 8, Essential 6, and Stempro34.

[0035] In another embodiment of the present invention, the differentiation medium comprises at least one selected from the group consisting of CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, FMS-like tyrosine kinase 3 ligand (FLT3L), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-15 (IL-15), and SB203580. May include, but is not limited to:

[0036] In another embodiment of the present invention, the differentiation medium comprises a first medium comprising Essential 8, CHIR-99021, BMP4, and VEGF;

[0037] A second medium containing Essential 6, SB431542, SCF, and VEGF;

[0038] Medium 3 containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, SCF, and FLT3L;

[0039] A fourth medium containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, and FLT3L; and

[0040] It may include at least one selected from the group consisting of Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, FLT3L, and a fifth medium comprising SB203580, but is not limited thereto.

[0041] As another embodiment of the present invention, the natural killer cells may express CD56 and CD45 and not express CD3, but are not limited thereto.

[0042] As another embodiment of the present invention, the medium for natural killer cell expansion comprises Stempro34, GlutaMAX, and Primosin; and

[0043] An antioxidant cocktail may include, but is not limited to, one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-15 (IL-15), interleukin-18 (IL-18), and interleukin-21 (IL-21).

[0044] In another embodiment of the present invention, when the natural killer cells are differentiated from non-genetically modified human induced pluripotent stem cells, the medium for natural killer cell expansion comprises Stempro34, Glutamax, Primosin, antioxidant cocktail, IL-2, and IL-15.

[0045] When the natural killer cells are differentiated from genetically engineered human induced pluripotent stem cells, the medium for natural killer cell expansion may include Stempro34, Glutamax, Primosin, antioxidant cocktail, IL-2, IL-15, and IL-18, or may include Stempro34, Glutamax, Primosin, antioxidant cocktail, IL-2, IL-15, IL-18, and IL-21, but is not limited thereto.

[0046] In another embodiment of the present invention, when the natural killer cells are differentiated from non-genetically modified human induced pluripotent stem cells, the medium for natural killer cell expansion contains IL-2 in an amount of 10 to 1000 IU and IL-15 in an amount of 5 to 50 ng / ml.

[0047] When the above natural killer cells are differentiated from genetically engineered human induced pluripotent stem cells, the medium for natural killer cell expansion may contain IL-2 at 10 to 1000 IU, IL-15 at 5 to 50 ng / ml, IL-18 at 50 to 250 ng / ml, and IL-21 at 1 to 20 ng / ml, but is not limited thereto.

[0048] As another embodiment of the present invention, the step of culturing in the medium for natural killer cell expansion may be performed within 7 to 28 days, but is not limited thereto.

[0049] As another embodiment of the present invention, the cell number ratio of the feeder cells and natural killer cells may be 0.5 to 5:1, but is not limited thereto.

[0050] In addition, the present invention provides a composition for expanding natural killer cells, which comprises the feeder cells of the present invention as an effective ingredient.

[0051] In addition, the present invention provides a kit for natural killer cell expansion, which comprises the feeder cells of the present invention, a differentiation medium, and a natural killer cell expansion medium as active ingredients.

[0052] In addition, the present invention provides a use of a composition comprising the feeder cell of the present invention as an active ingredient for expanding natural killer cells.

[0053] The present invention also provides a use for the preparation of a preparation for natural killer cell expansion comprising the feeder cells of the present invention as an active ingredient.

[0054]

[0055] When feeder cells containing the dual plasmid including the mb4-1BBL and mbIL-21 genes according to the present invention were co-cultured at a specific ratio in a medium for expanding natural killer cells containing cytokines such as interleukin-18 after differentiation of human induced pluripotent stem cells into natural killer cells, it was confirmed that the proliferation of induced NK cells (iNK) increased. Therefore, it is expected that the natural killer cell expansion method according to the present invention can be used to mass-produce natural killer cells differentiated from human induced pluripotent stem cells and easily utilize it for the development of various immune disease and cancer treatments.

[0056]

[0057] Figure 1 is a diagram schematically illustrating a differentiation process from human induced pluripotent stem cells into natural killer cells according to one embodiment of the present invention.

[0058] FIG. 2 is a drawing showing the structure of a mb4-1BBL and mbIL-21 dual plasmid vector according to one embodiment of the present invention.

[0059] FIG. 3 is a diagram schematically illustrating a process for selecting K562 feeder cells into which mb4-1BBL and mbIL-21 dual plasmids have been introduced according to one embodiment of the present invention.

[0060] FIG. 4a is a diagram showing the results of confirming the expression of mb4-1BBL and mbIL-21 according to the culture period in K562 feeder cells into which mb4-1BBL and mbIL-21 dual plasmids were introduced according to one embodiment of the present invention, using flow cytometry (FACS).

[0061] FIGS. 4b and 4c are drawings showing the results of flow cytometry analysis of mb4-1BBL and mbIL-21 expression before and after MMC treatment in K562 feeder cells into which mb4-1BBL and mbIL-21 dual plasmids were introduced according to one embodiment of the present invention.

[0062] FIG. 5a is a drawing showing the results of confirming morphological changes according to the ratio of K562 feeder cells and iNK cells introduced with mb4-1BBL and mbIL-21 dual plasmids during expansion of iNK cells according to one embodiment of the present invention.

[0063] FIG. 5b is a diagram showing the results of confirming the number of proliferated cells according to the ratio of K562 feeder cells and iNK cells into which mb4-1BBL and mbIL-21 dual plasmids have been introduced during the expansion of iNK cells according to one embodiment of the present invention.

[0064] FIGS. 5c to 5e are diagrams showing the results of confirming the marker expression of cells according to the ratio of K562 feeder cells and iNK cells into which mb4-1BBL and mbIL-21 dual plasmids have been introduced during the expansion of iNK cells according to one embodiment of the present invention.

[0065] FIGS. 6A and 6B are drawings showing the results of confirming morphological changes according to the ratio of genetically engineered iNK cells and cytokine combinations and K562 feeder cells into which mb4-1BBL and mbIL-21 dual plasmids are introduced during expansion of genetically engineered iNK cells according to one embodiment of the present invention. FIG. 6A is a drawing showing a case where the ratio of feeder cells (aAPC):iNK is 2:1, and FIG. 6B is a drawing showing a case where the ratio of aAPC:iNK is 3:1.

[0066] FIG. 6c is a diagram showing the results of confirming the number of proliferated cells according to the ratio of genetically engineered iNK cells and K562 feeder cells into which mb4-1BBL and mbIL-21 dual plasmids were introduced and the cytokine combination during expansion of genetically engineered iNK cells according to one embodiment of the present invention.

[0067] FIG. 6d is a diagram showing the results of confirming the ratio of genetically engineered iNK cells to K562 feeder cells introduced with mb4-1BBL and mbIL-21 dual plasmids and cell survival rate according to cytokine combination during expansion of genetically engineered iNK cells according to one embodiment of the present invention.

[0068] FIGS. 6E and 6F are drawings showing the results of confirming the ratio of genetically engineered iNK cells to K562 feeder cells introduced with mb4-1BBL and mbIL-21 dual plasmids and the expression of cell markers (CD45, CD3, CD16, NKG2A, NKG2D, CAR, CD24) according to the cytokine combination during the expansion of genetically engineered iNK cells according to one embodiment of the present invention.

[0069] FIGS. 7A to 7D are drawings showing the results of flow cytometry analysis to confirm the expression of cell markers (CD45, CD3, CD16, NKG2A, NKG2D) according to cytokine combinations when the ratio of K562 feeder cells introduced with mb4-1BBL and mbIL-21 dual plasmids and genetically engineered iNK cells is 2:1 during expansion of genetically engineered iNK cells according to one embodiment of the present invention.

[0070] FIGS. 7E to 7H are drawings showing the results of flow cytometry analysis to confirm the expression of cell markers (CD45, CD3, CD16, NKG2A, NKG2D) according to cytokine combinations when the ratio of K562 feeder cells introduced with mb4-1BBL and mbIL-21 dual plasmids and genetically engineered iNK cells is 3:1 during expansion of genetically engineered iNK cells according to one embodiment of the present invention.

[0071]

[0072] In one experimental example of the present invention, a dual plasmid vector was prepared to express mb4-1BBL and mbIL-21, and it was confirmed that the expression of mb4-1BBL and mbIL-21 was maintained for up to 60 days after culture in K562 feeder cells introduced therein (see Experimental Example 1).

[0073] In another experimental example of the present invention, when the cell proliferation was confirmed according to the ratio of feeder cells and iNK cells during iNK cell expansion, it was confirmed that when the ratio of K562 feeder cells and iNK cells introduced with dual plasmids expressing mb4-1BBL and mbIL-21 was 2:1, a higher cell proliferation rate was shown compared to the ratios of 0.5:1 and 1:1, and the expression of CD45, an NK cell marker, was all high, the expression of CD3, a T cell marker, was hardly expressed, the expression of NKG2A, an NK cell suppression marker, was decreased, and the expression of NKG2D, an activation marker, was similar (see Experimental Example 2).

[0074] In another experimental example of the present invention, when expanding genetically engineered iNK cells, cell proliferation was confirmed according to the ratio of feeder cells and iNK cells and the combination of cytokines. As a result, it was confirmed that when the ratio of K562 feeder cells introducing dual plasmids expressing mb4-1BBL and mbIL-21 to genetically engineered iNK cells was 3:1 and the cytokines were a combination of IL2, IL15, IL21, and IL18, a high cell proliferation rate was observed. In addition, when IL18 was present among the cytokines, cell viability increased, and when the ratio of feeder cells (aAPC):iNK was 3:1 and the cytokines were a combination of IL2, IL15, IL21, and IL18, the expression of NK cell activation markers CD16 and NKG2D increased, and the expression of the suppression marker NKG2A decreased (see Experimental Example 3).

[0075]

[0076] Hereinafter, the present invention will be described in detail.

[0077]

[0078] The present invention provides a dual plasmid vector comprising a membrane bound human 4-1BB ligand (mb4-1BBL) and a membrane bound human interleukin-21 (mbIL-21) gene.

[0079] In the present invention, “dual plasmid vector” means a vector manufactured by inserting two or more genes into one plasmid.

[0080] In the present invention, a "vector" refers to any medium for cloning and / or transferring nucleotides into a host cell. A vector may be a replicating unit (replicon) capable of binding to another DNA fragment and causing replication of the bound fragment. A "replicating unit" refers to any genetic unit (e.g., a plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., is capable of replicating under its own control. The term "vector" encompasses viral and non-viral vectors for introducing nucleotides into a host cell in vitro, ex vivo, or in vivo.

[0081] In the present invention, the “4-1BB ligand (4-1BBL)” is also called TNFSF9 (Tumor Necrosis Factor Superfamily Member 9) and is a co-stimulatory ligand that binds to the 4-1BB (or CD137) receptor to activate T cells and NK cells. 4-1BBL is mainly expressed in immune cells (T cells, dendritic cells, NK cells) and plays a role in promoting the survival and proliferation of T cells and NK cells. In the present invention, the 4-1BBL may be a cell membrane-bound human 4-1BBL (mb4-1BBL), and the mb4-1BBL gene may be composed of or include a base sequence having a sequence homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% with the base sequence of SEQ ID NO: 1, but is not limited thereto.

[0082] In the present invention, the “interleukin-21 (IL-21)” is an important cytokine that regulates the proliferation and function of immune cells (T cells, NK cells, B cells, etc.), and in particular, plays a role in promoting anti-cancer immune responses by activating NK cells and CD8+ T cells. In the present invention, the IL-21 included in the vector may be cell membrane-bound human IL-21 (mbIL-21), and the mbIL-21 gene may be composed of or include a base sequence having a sequence homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% to the base sequence of SEQ ID NO: 2, but is not limited thereto.

[0083] In the present invention, the vector is mEF1α (Mouse elongation factor 1α), Neo r (neomycin resistance gene), and rEF1α (Rat elongation factor 1α), but is not limited thereto. In this case, the mEF1α, Neo r , and rEF1α genes known in the art can be used, and are not limited to their sequences.

[0084] In the present invention, the vector may have the structure of Fig. 2. As one embodiment of the present invention, the vector may have the structure of mEF1α->mbIL-21->rEF1α->mb4-1BBL->Neo. r It may proceed in the direction of, but is not limited to, this.

[0085]

[0086] In addition, the present invention provides a feeder cell transfected with the dual plasmid vector.

[0087] In the present invention, “transfection” is also called transfection and is a compound word of “trans” and “infection” and refers to injecting nucleic acid into a eukaryotic cell. In the present invention, the method of transfection may use known methods such as nucleofection, transient transfection, microinjection, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, and electroporation, but is not limited thereto.

[0088] In the present invention, the feeder cell may be a K562 cell, but is not limited thereto.

[0089] In the present invention, the feeder cells may include, but are not limited to, cells in which human leukocyte antigen (HLA) expression is inhibited.

[0090]

[0091] In addition, the present invention provides a method for differentiating stem cells into natural killer cells, comprising a step of culturing isolated stem cells in a differentiation medium.

[0092] In addition, the present invention comprises a step of culturing separated stem cells in a differentiation medium to differentiate them into natural killer cells; and

[0093] A method for expanding natural killer cells is provided, comprising a step of co-culturing the natural killer cells with the feeder cells in a medium for expanding natural killer cells.

[0094] In the present invention, the stem cells may be human induced pluripotent stem cells (iPSCs), but are not limited thereto.

[0095] In the present invention, "induced pluripotent stem cells (iPSCs)" refer to cells that have been artificially induced to possess pluripotency through a process of artificially reprogramming differentiated cells. Since iPSCs can be obtained by reprogramming differentiated cells using reprogramming factors, they enable the generation of patient-compatible pluripotent cell lines without somatic cell nuclear transfer. Therefore, iPSCs can be derived from patient cells, thereby avoiding immune rejection when applied clinically. Furthermore, since iPSCs do not use eggs or embryos, they have the advantage of being free of bioethical controversies and religious criticism.

[0096] In the present invention, the human induced pluripotent stem cell may be a non-genetically modified human induced pluripotent stem cell or a genetically modified human induced pluripotent stem cell, but is not limited thereto.

[0097] In the present invention, the genetically engineered human induced pluripotent stem cell may be a human induced pluripotent stem cell into which a chimeric antigen receptor (CAR) gene has been introduced, but is not limited thereto.

[0098] In the present invention, "chimeric antigen receptor (CAR)" refers to synthetic receptors capable of targeting a specific antigen, and the CAR according to the present invention may target the human CD19 antigen. In the present invention, the CAR may use a CD19 CAR known in the art, and is not limited to its sequence as long as it targets the human CD19 antigen.

[0099] In the present invention, the CAR may include, but is not limited to, one or more selected from the group consisting of an antigen-binding domain, a hinge region, a transmembrane domain (TM), and an intracellular signaling domain (cytoplasmic domain or cytoplasmic signaling domain).

[0100] In the present invention, "antigen-binding domain" refers to an antibody, protein, or peptide domain capable of specifically recognizing and binding to a target antigen. In the present invention, "antigen" refers to a polypeptide, compound, or other substance capable of specifically binding to a humoral immune mediator such as an antibody or a cellular immune mediator such as a T cell receptor. In the present invention, the target antigen may be the human CD19 antigen.

[0101] The antigen binding domain may be an antigen-specific antibody, an antigen-specific protein, an antigen-specific peptide, or a fragment of an antibody (preferably, an antigen-binding fragment).

[0102] In the present invention, "antibody" means an immunoglobulin molecule that immunologically reacts with a specific antigen (epitope), and includes polyclonal antibodies, monoclonal antibodies, and functional fragments thereof. In addition, the term may include forms produced by genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous antibodies (e.g., bispecific antibodies). The antibody comprises a variable region of a heavy chain and / or a light chain (V H , heavy chain variable region; V L , light chain variable region). The variable region includes a portion forming an antigen-binding site of an antibody molecule as a primary structure, and the antibody of the present invention may be composed of a fragment including the variable region. A linker may be positioned between the light chain and heavy chain variable regions of the antibody, protein, peptide, or antigen-binding fragment of the antibody. The “linker” refers to a polypeptide that connects the light chain variable region and the heavy chain variable region to each other without damaging their original antigen-binding properties.

[0103] In the present invention, the "hinge region" refers to a region located between the antigen-binding domain and the transmembrane domain and functions as a flexible linker. The hinge region ensures that the antigen-binding domain is properly positioned when bound to an antigen, thereby forming a stable bond with the antigen. For example, the hinge region serves the purpose of extending the antigen-binding domain from the cell membrane of a cell expressing a CAR.

[0104] In the present invention, the term "transmembrane domain (TM)" refers to any polypeptide or oligopeptide that functions to cross the cell membrane and connect extracellular and intracellular signaling domains. The transmembrane domain can penetrate the cell membrane so that the antigen-binding domain of the chimeric antigen receptor can be located on the cell surface (outside the cell) and the intracellular signaling domain can be located within the cell. In other words, the transmembrane domain serves as a support for the chimeric antigen receptor while simultaneously connecting the antigen-binding domain (or hinge domain) and the intracellular signaling domain.

[0105] In the present invention, the "intracellular signaling domain (cytoplasmic domain or cytoplasmic signaling domain)" refers to a polypeptide or oligopeptide located inside the cell membrane. The intracellular signaling domain receives a signal transmitted by the antigen binding domain and transmits the signal to the inside of a cell expressing the CAR. The signal is transmitted inside the cell and causes activation or inhibition of a biological process. The intracellular signaling domain is not particularly limited in type as long as it transmits a signal capable of inducing cell activation when the antigen binding domain binds to an antigen. In the present invention, the intracellular signaling domain may include one or more intracellular co-stimulatory domains. The co-stimulatory domain is located in the extracellular or intracellular portion of the CAR and serves to transmit a signal to a cell expressing the CAR. That is, the co-stimulatory domain contributes to inducing a sufficient response (activation) of a T cell following binding of a target antigen. The co-stimulatory domain may be selected from, but is not limited to, domains derived from, for example, CD27, CD28, 41BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1 (lymphocyte function-associated antigen-1), CD2, CD7, LIGHT, NKG2C, and / or B7-H3.

[0106] In the present invention, the differentiation medium may include one or more media selected from the group consisting of Essential 8, Essential 6, and Stempro34, but is not limited thereto.

[0107] In the present invention, the differentiation medium may further include at least one selected from the group consisting of CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, FMS-like tyrosine kinase 3 ligand (FLT3L), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-15 (IL-15), and SB203580. However, it is not limited to this.

[0108] In the present invention, the differentiation medium is a first medium containing Essential 8, CHIR-99021, BMP4, and VEGF;

[0109] A second medium containing Essential 6, SB431542, SCF, and VEGF;

[0110] Medium 3 containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, SCF, and FLT3L;

[0111] A fourth medium containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, and FLT3L; and

[0112] It may include at least one selected from the group consisting of Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, FLT3L, and a fifth medium comprising SB203580, but is not limited thereto.

[0113] In the present invention, the step of culturing the separated stem cells in a differentiation medium may include, but is not limited to, a step of culturing in a first medium (differentiation media 1 of Example 2-2-2), a step of culturing in a second medium (differentiation media 2 of Example 2-2-2), a step of culturing in a third medium (differentiation media 3 of Example 2-2-2), a step of culturing in a fourth medium (differentiation media 4-1 of Example 2-2-2), and a step of culturing in a fifth medium (differentiation media 4-2 of Example 2-2-2).

[0114] According to one embodiment of the present invention, induced pluripotent stem cells can be differentiated into natural killer cells through the step of culturing in the first to fifth media.

[0115] In the present invention, the CHIR-99021, SB431542, or beta-mercaptoethanol may be included in the differentiation medium at, but is not limited to, 0.1 to 5 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.3 to 5 μM, 0.3 to 3 μM, 0.3 to 2 μM, 0.5 to 5 μM, 0.5 to 3 μM, or 0.5 to 2 μM.

[0116] In the present invention, the BMP4 or VEGF may be included in the differentiation medium at 10 to 200 ng / ml, 10 to 150 ng / ml, 10 to 100 ng / ml, 50 to 200 ng / ml, 50 to 150 ng / ml, 50 to 100 ng / ml, 60 to 200 ng / ml, 60 to 150 ng / ml, or 60 to 100 ng / ml, but is not limited thereto.

[0117] In the present invention, the SCF or FLT3L may be included in the differentiation media at 1 to 100 ng / ml, 1 to 70 ng / ml, 1 to 50 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 5 to 100 ng / ml, 5 to 70 ng / ml, 5 to 50 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, 10 to 100 ng / ml, 10 to 70 ng / ml, 10 to 50 ng / ml, 10 to 30 ng / ml, 10 to 20 ng / ml, 20 to 100 ng / ml, 20 to 70 ng / ml, or 20 to 50 ng / ml, but is not limited thereto.

[0118] In the present invention, the sodium selenite may be included in the medium at 0.1 to 20 ng / ml, 0.1 to 10 ng / ml, 0.1 to 5 ng / ml, 0.5 to 20 ng / ml, 0.5 to 10 ng / ml, 0.5 to 5 ng / ml, 1 to 20 ng / ml, 1 to 10 ng / ml, or 1 to 5 ng / ml, but is not limited thereto.

[0119] In the present invention, the ethanolamine may be included in the medium at 1 to 80 μM, 1 to 70 μM, 1 to 60 μM, 10 to 80 μM, 10 to 70 μM, 10 to 60 μM, 30 to 80 μM, 30 to 70 μM, or 30 to 60 μM, but is not limited thereto.

[0120] In the present invention, the ascorbic acid may be L-ascorbic acid, and may be included in the medium at 1 to 40 mg / L, 1 to 30 mg / L, 1 to 20 mg / L, 5 to 40 mg / L, 5 to 30 mg / L, 5 to 20 mg / L, 10 to 40 mg / L, 10 to 30 mg / L, or 10 to 20 mg / L, but is not limited thereto.

[0121] In the present invention, the IL-3 may be included in the medium at 0.1 to 10 ng / ml, 0.1 to 7 ng / ml, 0.1 to 5 ng / ml, 1 to 10 ng / ml, 1 to 7 ng / ml, 1 to 5 ng / ml, 2 to 10 ng / ml, 2 to 7 ng / ml, or 2 to 5 ng / ml, but is not limited thereto.

[0122] In the present invention, the IL-7 may be included in the medium at 1 to 50 ng / ml, 1 to 40 ng / ml, 1 to 30 ng / ml, 5 to 50 ng / ml, 5 to 40 ng / ml, 5 to 30 ng / ml, 10 to 50 ng / ml, 10 to 40 ng / ml, or 10 to 30 ng / ml, but is not limited thereto.

[0123] In the present invention, the IL-15 may be included in the medium at 1 to 40 ng / ml, 1 to 30 ng / ml, 1 to 20 ng / ml, 3 to 40 ng / ml, 3 to 30 ng / ml, 3 to 20 ng / ml, 5 to 40 ng / ml, 5 to 30 ng / ml, or 5 to 20 ng / ml, but is not limited thereto.

[0124] In the present invention, the SB203580 may be included in the medium at 0.1 to 10 μM, 0.1 to 7 μM, 0.1 to 5 μM, 0.5 to 10 μM, 0.5 to 7 μM, 0.5 to 5 μM, 1 to 10 μM, 1 to 7 μM, or 1 to 5 μM, but is not limited thereto.

[0125] In the present invention, the step of culturing the separated stem cells in a differentiation medium can be performed within 46 to 60 days, 46 to 58 days, 46 to 56 days, 46 to 54 days, or 46 to 52 days, and the culturing at each step can be performed for 1 to 26 days, 1 to 22 days, 1 to 18 days, 1 to 14 days, 1 to 10 days, 1 to 6 days, or 1 to 2 days, but is not limited thereto.

[0126] In the present invention, the differentiated natural killer cells may express CD56 and CD45 and not express CD3, but are not limited thereto.

[0127] In the present invention, induced natural killer cells (induced-NK cells, iNK) are NK cells derived from induced pluripotent stem cells, and may refer to natural killer cells differentiated from induced pluripotent stem cells.

[0128] In the present invention, “expansion” means a process of proliferation or scale-up of cells, and “expansion of natural killer cells” may mean a process of mass-producing initially differentiated NK cells (iNK) from induced pluripotent stem cells so that they can be used for experiments or treatments.

[0129] When using the method for expanding natural killer cells according to the present invention, the number of natural killer cells can be increased by 5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 110 times or more, 120 times or more, 130 times or more, 5 to 140 times, 10 to 140 times, 20 to 140 times, 50 to 140 times, 70 to 140 times, 100 to 140 times, 120 to 140 times, or 130 to 140 times, but is not limited thereto.

[0130] In the present invention, the natural killer cell expansion medium is Stempro34, GlutaMAX, and Primosin; and

[0131] An antioxidant cocktail may include, but is not limited to, one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-15 (IL-15), interleukin-18 (IL-18), and interleukin-21 (IL-21).

[0132] In the present invention, a supplement may be added to Stempro34 in the medium for natural killer cell expansion, and the supplement may include at least one selected from the group consisting of beta-mercaptoethanol, sodium selenite, ethanolamine, and ascorbic acid, but is not limited thereto.

[0133] In the present invention, the medium for natural killer cell expansion may include, but is not limited to, Stempro34, Glutamax, Primosin, antioxidant cocktail, IL-2, and IL-15.

[0134] In the present invention, the medium for natural killer cell expansion may include, but is not limited to, Stempro34, Glutamax, Primosin, antioxidant cocktail, IL-2, IL-15, and IL-18.

[0135] In the present invention, the medium for natural killer cell expansion may include, but is not limited to, Stempro34, Glutamax, Primosin, antioxidant cocktail, IL-2, IL-15, IL-18, and IL-21.

[0136] In the present invention, the IL-2 may be included in the medium for natural killer cell expansion at 10 to 1000 IU, but is not limited thereto.

[0137] In the present invention, the IL-15 may be included in the medium for natural killer cell expansion at 5 to 50 ng / ml, but is not limited thereto.

[0138] In the present invention, the IL-18 may be included in the medium for natural killer cell expansion at 50 to 250 ng / ml, but is not limited thereto.

[0139] In the present invention, the IL-21 may be included in the medium for natural killer cell expansion at 1 to 20 ng / ml, but is not limited thereto.

[0140] According to one embodiment of the present invention, in the case of natural killer cells differentiated from non-genetically modified human induced pluripotent stem cells, the natural killer cells can be expanded by culturing them in a natural killer cell expansion medium containing Stempro34, Glutamax, and Primosin, an antioxidant cocktail, IL-2, and IL-15, and in the case of natural killer cells differentiated from genetically modified human induced pluripotent stem cells, the natural killer cells can be expanded by culturing them in a natural killer cell expansion medium containing Stempro34, Glutamax, and Primosin, an antioxidant cocktail, IL-2, IL-15, and IL-18, or Stempro34, Glutamax, and Primosin, an antioxidant cocktail, IL-2, IL-15, IL-18, and IL-21, but is not limited thereto.

[0141] In the present invention, in the case of natural killer cells differentiated from non-genetically modified human induced pluripotent stem cells, IL-2 may be included in an amount of 10 to 1000 IU and IL-15 may be included in an amount of 5 to 50 ng / ml in the medium for natural killer cell expansion.

[0142] In the case of natural killer cells differentiated from genetically engineered human induced pluripotent stem cells, the medium for natural killer cell expansion may contain, but is not limited to, IL-2 at 10 to 1000 IU, IL-15 at 5 to 50 ng / ml, IL-18 at 50 to 250 ng / ml, and IL-21 at 1 to 20 ng / ml.

[0143] In the present invention, the step of culturing in the natural killer cell expansion medium may be performed within 7 to 28 days, 7 to 25 days, 7 to 22 days, 7 to 18 days, or 7 to 14 days, but is not limited thereto.

[0144] In the present invention, the cell number ratio of the feeder cells and natural killer cells may be, but is not limited to, 0.5 to 5:1, 0.5 to 4:1, 0.5 to 3:1, 0.5 to 2:1, 0.5 to 1:1, 1 to 5:1, 1 to 4:1, 1 to 3:1, 1 to 2:1, 2 to 5:1, 2 to 4:1, 2 to 3:1, 3 to 5:1, 3 to 4:1, 2:1, or 3:1. In this case, the natural killer cells may include both non-genetically modified human induced pluripotent stem cells and natural killer cells differentiated from genetically modified human induced pluripotent stem cells.

[0145] In addition, the present invention provides a composition for expanding natural killer cells, which comprises the feeder cells of the present invention as an effective ingredient.

[0146] In addition, the present invention provides a kit for natural killer cell expansion, which comprises the feeder cells of the present invention, a differentiation medium, and a natural killer cell expansion medium as active ingredients.

[0147] In the present invention, the “kit” refers to a tool that enables the expansion of natural killer cells by including the feeder cells, differentiation medium, and natural killer cell expansion medium. The kit of the present invention may include, in addition to the feeder cells, differentiation medium, and natural killer cell expansion medium, other components, compositions, solutions, devices, etc. that are typically required for the expansion of natural killer cells. At this time, the feeder cells, differentiation medium, and natural killer cell expansion medium may be applied one or more times without limitation, and there is no limitation on the order in which each substance is applied, and the application of each substance may be performed simultaneously or microscopically.

[0148] In the present invention, the kit may include a container; an instruction manual; and the feeder cells, the differentiation medium, and the natural killer cell expansion medium. The container may serve to package the feeder cells, the differentiation medium, and the natural killer cell expansion medium, and may also serve to store and fix the feeder cells, the differentiation medium, and the natural killer cell expansion medium. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., and these may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be initially equipped with a completely or partially detachable stopper that is part of the container or can be mechanically, adhesively, or otherwise attached to the container, and may also be equipped with a stopper that allows access to the contents by a syringe needle. The kit may include an outer package, and the outer package may include instructions regarding the use of the components.

[0149]

[0150] In addition, the present invention provides a use of a composition comprising the feeder cell of the present invention as an active ingredient for expanding natural killer cells.

[0151] The present invention also provides a use for the preparation of a preparation for natural killer cell expansion comprising the feeder cells of the present invention as an active ingredient.

[0152]

[0153] Hereinafter, preferred examples and experimental examples are presented to aid in understanding the present invention. However, the following examples and experimental examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples and experimental examples.

[0154]

[0155] [Example]

[0156] Example 1. Human induced pluripotent stem cell culture

[0157] 1-1. Experimental Preparation

[0158] First, for culture vessel coating, 1.5 ml of DPBS containing 9.6 μl of iMatrix-511 (175 μg) per well was added to a 6-well plate, and coating was performed in a coating incubator for 1 hour, and subculture was performed after 1 hour.

[0159] Instead of warming the culture medium to 37°C using StemFit Basic04, the medium was thawed at room temperature, and 1 ml of the antibiotic Primosin was added to the medium and mixed to prepare a complete medium.

[0160] To prepare a Y-27632 solution, Y-27632 was added to distilled water (DW) to make a 10 mM solution. This was then aliquoted into EP tubes and stored at -20°C.

[0161]

[0162] 1-2. Human induced pluripotent stem cell culture

[0163] The complete medium prepared in Example 1-1 above was prepared by warming it at room temperature for more than 30 minutes.

[0164] The wild-type (WT) and genetically engineered iPSCs (upCAR-iPSCs) used in the present invention were produced by direct reprogramming. UpCAR-iPSCs were iPSCs into which a CAR targeting CD19 (CAR(CD19)) and the CD24 gene were introduced.

[0165]

[0166] 1-2-1. General cultivation

[0167] Induced pluripotent stem cells were seeded at 1.5x10 per well in a 6-well plate. 4Cells were seeded and cultured in an incubator at 37°C and 5% CO2. After 24 hours, the complete medium was removed and replaced with 2.5 ml of fresh complete medium, and cultured until the next subculture.

[0168]

[0169] 1-2-2. Subculture

[0170] For subculture, 10 mM Y-27632 prepared in Example 1-1 above was dissolved at room temperature and added 1000x to the complete medium warmed at room temperature, and the culture medium of the plate containing the cells to be subcultured was removed. Then, 1 mL of DPBS was added per well of a 6-well plate and washed. 1 ml of TrypLE reagent was added and incubated for 15 minutes at 37°C, 5% CO2 conditions. After confirming that the cells had detached, 1 ml of the reacted TrypLE reagent was added to a 15 ml tube containing 8 ml of complete medium, and the medium was added once more to the existing 15 ml tube (8 ml of complete medium + TrypLE reagent), and the cells were collected again and placed into the 15 ml tube. Then, this was centrifuged at 300 g for 5 minutes, and the medium was removed after leaving only the cells. After that, add 3 ml of medium + Y-27632 and count the cells, and 3x10 for 6 wells. 4 Cells were seeded at 2.5 ml / well.

[0171]

[0172] Example 2. Differentiation of human induced pluripotent stem cells into natural killer cells.

[0173] 2-1. Experimental Preparation

[0174] First, for culture vessel coating, 75 μl of iMatrix-511 (175 μg) was added to 15 ml of DPBS in a T-75 flask and coated for 1 hour in a coating incubator.

[0175] Instead of warming the culture medium to 37°C using StemFit Basic04, the medium was melted at room temperature, and 1 ml of the antibiotic Primosin was added to the medium and mixed to prepare a complete growth medium.

[0176] To prepare a Y-27632 solution, Y-27632 was added to distilled water (DW) to make a 10 mM solution. This was then aliquoted into EP tubes and stored at -20°C.

[0177]

[0178] 2-2. Differentiation of human induced pluripotent stem cells into natural killer cells

[0179] The proliferation complete medium prepared in Example 2-1 above was prepared by warming it at room temperature for more than 30 minutes.

[0180]

[0181] 2-2-1. Spheroid manufacturing and culture

[0182] After seeding the human induced pluripotent stem cells cultured in Example 1 on a SPHERICAL PLATE 5D plate, spheroids were produced by culturing them for 24 hours at 37°C and 5% CO2 conditions.

[0183] Then, the manufactured spheroids were seeded into T-75 flasks coated in Example 2-1, and 15 ml of the complete proliferation medium prepared in Example 2-1 and warmed at room temperature for more than 30 minutes was added to each T-75 flask, and seeded into 7 flasks. Thereafter, general culture was performed in the same manner as in Example 1-2-1 for 3 to 5 days until the colony size reached 750 μm to 1000 μm.

[0184]

[0185] 2-2-2. Differentiation process into natural killer cells

[0186] In the above Example 2-2-1, spheroids manufactured and cultured using human induced pluripotent stem cells were differentiated into natural killer cells through the following process, and this process is schematically shown in Figure 1.

[0187] Day 0: After culturing spheroids in a T-75 flask in Example 2-2-1, the growth medium (Stemfit Basic04) was removed by suction, washed with Essential 8 medium, and cultured by adding 15 ml of differentiation media 1 (Essential 8 + 0.5~2 μM CHIR-99021 + 60~100 ng / ml BMP4 + 60~100 ng / ml VEGF).

[0188] Day 2~3: After aspirating and removing differentiation media 1, 15 ml of differentiation media 2 (Essential 6 + 0.5~2 μM SB431542 + 20~50 ng / ml SCF + 60~100 ng / ml VEGF) was added and cultured.

[0189] Day 4~5: After aspirating and removing differentiation media 2, 15 ml of differentiation media 3 (stempro-34 + 0.5~2 μM β-mercaptoethanol, 1~5 ng / ml sodium selenite, 30~60 μM ethanolamine, 10~20 mg / L ascorbic acid, 20~50 ng / ml SCF, 20~50 ng / ml FLT-3L) was added and cultured.

[0190] Day 6~7: Add 15 ml of differentiation media 3 (total 30 ml).

[0191] Days 8–10: After half the medium was changed, 15 ml of the medium in the flask was harvested and placed in a 50 ml tube. The medium was then centrifuged at 300 g for 5 minutes, the medium was removed, and Differentiation Media 3 was added to the differentiating flask and cultured.

[0192] Day 10~14: The medium in the differentiating T-75 flask was harvested (30 ml / T-75), centrifuged at 300 g for 5 min, and then 15 ml of differentiation media 4-1 (stempro-34 + 0.5~2 μM β-mercaptoethanol, 1~5 ng / ml sodium selenite, 30~60 μM ethanolamine, 10~20 mg / L ascorbic acid, 2~5 ng / ml IL-3, 10~30 ng / ml SCF, 10~30 ng / ml IL-7, 5~20 ng / ml IL-15, 5~20 ng / ml FLT-3L) was added (15 ml / T-75).

[0193] Day 12~16: Add 15 ml of differentiation media 4-1 (total 30 ml).

[0194] Day 16~20: Complete change was made from differentiation media 4-1 to differentiation media excluding IL-3, then centrifuged at 300 g for 5 minutes and 15 ml of differentiation media 4-2 was added.

[0195] Day 18~22: Add 15 ml of differentiation media 4-2.

[0196] Days 26–48: Half the medium was changed every four days. Then, 15 ml per T-75 flask was placed in a 50-ml tube and centrifuged at 300 g for 5 minutes. The medium was removed, and 15 ml of fresh Differentiation Media 4-2 was added to suspend the cells and placed in the flasks.

[0197] Days 46–52: Differentiation was completed, and cells were harvested for various analyses. The medium in the flask was collected and transferred to a 50-ml tube. The tube was centrifuged at 300 g for 5 minutes, then Differentiation Media 4-2 was added and the cells were counted (using a cell counter, not manual counting). The cell counts for each analysis were then determined.

[0198]

[0199] 2-3. Verification of differentiation of human induced pluripotent stem cells into natural killer cells using flow cytometry (FACS).

[0200] After harvesting the differentiating cells at each stage, they were centrifuged at 300 g for 5 minutes. Afterwards, the medium was removed, 10 ml of FACS buffer was added, and the cells were washed by centrifugation at 300 g for 5 minutes. Then, 1x10 5 Cells were added, and 1 μl of antibodies and isotypes for each marker in the differentiation verification process into natural killer cells shown in Table 1 below were added, and the mixture was incubated on ice for 1 hour. Information on the antibodies used was analyzed according to the analysis day, referring to Table 1.

[0201] Afterwards, 1 ml of FACS buffer was added and centrifuged at 300 g for 5 minutes to wash, and the process of adding 1 ml of FACS buffer and centrifuging at 300 g for 5 minutes was repeated 3 times. Then, measurements were made using a FACS device.

[0202]

[0203] Example 3. Production of genetically engineered feeder cells

[0204] 3-1. Preparation of culture medium

[0205] 1 ml of the antibiotic primocin was added to RPMI 1640 medium, mixed, and FBS was added at a ratio of 10% to prepare complete medium.

[0206]

[0207] 3-2. K562 cell culture

[0208] The complete medium prepared in Example 3-1 above was prepared by warming it in an incubator at 37°C and 5% CO2 for more than 30 minutes.

[0209] For subculture, the culture medium from the flask containing K562 cells to be subcultured was placed in a 50 ml tube, centrifuged at 300 g for 5 minutes, and the medium was removed after leaving only the cells. Then, the cells were resuspended in warmed complete medium. After counting the number of cells, 3x10 5 Cells were seeded in T25 flasks at a concentration of 10 cells / ml.

[0210]

[0211] 3-3. Introduction of mb4-1BBL and mbIL-21 dual plasmids into K562 cells.

[0212] 3-3-1. Preparation of culture medium

[0213] 1 ml of the antibiotic primocin was added to RPMI 1640 medium, mixed, and FBS was added at a ratio of 10% to prepare complete medium.

[0214]

[0215] 3-3-2. Harvesting K562 cells

[0216] The complete medium prepared in Example 3-3-1 above was prepared by warming it in an incubator at 37°C and 5% CO2 for more than 30 minutes.

[0217] To harvest K562 cells, the culture medium from the flask containing the cells to be introduced with the dual plasmid was placed in a 50 ml tube, centrifuged at 300 g for 5 minutes, and the medium was removed, leaving only the cells. The cells were then resuspended in DPBS, and the cell number was counted.

[0218]

[0219] 3-3-3. Electroporation

[0220] 4 ml of E buffer was added to the Neon electroporation kit tube and then mounted on the machine.

[0221] 1x10 K562 cells harvested in Example 3-3-2 above 6 After placing the cells in an EP tube, centrifugation was performed at 300 g for 5 minutes, washing was performed with 1 ml of DPBS, and centrifugation was performed again at 300 g for 5 minutes. The supernatant was removed, leaving only the cells.

[0222] Then, 10 μg of dual plasmid DNA containing the mb4-1BBL and mbIL-21 genes was added to the tube containing the cells, and resuspended in 100 μl of T buffer from the Neon electroporation kit.

[0223] At this time, the structure of the mb4-1BBL and mbIL-21 dual plasmid vector is shown in Fig. 2, and the nucleotide sequences of membrane bound (mb)4-1BBL and mbIL-21 inserted into the vector are shown in Table 2 below.

[0224]

[0225]

[0226] After attaching a 100 μl tip to the pipette of the Neon electroporation kit, cells and DNA in the T buffer were added, and 4 ml of E2 buffer was placed in a tube dedicated to electroporation and mounted on the machine. Then, the pipette was mounted on a tube containing E2 buffer and transfection was performed by electrical stimulation.

[0227] Afterwards, the cells were seeded in 6 wells and cultured in an incubator at 37°C and 5% CO2 for 3 days.

[0228]

[0229] 3-4. Selection of K562 cells with mb4-1BBL and mbIL-21 dual plasmids

[0230] K562 cells containing the dual plasmid transfected with the method of Example 3-3 above were selected through the following process, and this process is schematically shown in Figure 3.

[0231] Day 0: Selective medium 1 was prepared by adding 400 μg / ml G418 (50 μg / ml Geneticin) to 10% FBS RPMI medium. Then, the culture medium from the flask containing cells transfected with the mb4-1BBL plasmid was transferred to a 50 ml tube, centrifuged at 300 g for 5 min, and the medium was removed, leaving only the cells. Then, the cells were resuspended in DPBS, and the number of cells was counted. 3 x 10 5 Cells were seeded in 6-well plates at a concentration of 10 cells / ml and cultured, repeating at 3-4 day intervals for up to 14 days.

[0232] Day 14: After selection, cells were harvested using the method of Example 3-3-2 for FACS analysis.

[0233] After cell selection, culture was performed by adding G418 (working concentration 50 μg / ml geneticin).

[0234]

[0235] 3-5. Verification of K562 cells with dual plasmids using flow cytometry (FACS)

[0236] After harvesting the cells selected through the process of Example 3-4 above, they were centrifuged at 300 g for 5 minutes. Then, the medium was removed, 10 ml of FACS buffer was added, and the cells were washed by centrifugation at 300 g for 5 minutes, and 1x10 were placed in each FACS tube. 5 Cells were added. Then, 1 μl of each antibody and isotype was added to the tube and incubated on ice for 1 hour. The antibodies and isotypes used are as follows:

[0237] Isotype: PE Mouse IgG1, κIsotype (cat, 349043, BD Biosciences), APC Mouse IgG1, κIsotype Control (cat. 340442, BD).

[0238] Markers: PE Mouse anti-Human IL-21 (cat. 560463, BD Pharmingen™), CD137 Ligand (4-1BB Ligand) Monoclonal Antibody (4H3), APC (Cat. 17-5906-42, eBioscience™).

[0239] Afterwards, 1 ml of FACS buffer was added and centrifuged at 300 g for 5 minutes to wash, and the process of adding 1 ml of FACS buffer and centrifuging at 300 g for 5 minutes was repeated 3 times. Then, measurements were made using a FACS device.

[0240]

[0241] Example 4. Expansion of human induced pluripotent stem cells into natural killer cells.

[0242] 4-1. Preparation for iNK cell and upCAR-iNK cell expansion

[0243] 4-1-1. Preparation of basal medium

[0244] A supplement containing 0.5–2 μM β-mercaptoethanol, 1–5 ng / ml sodium selenite, 30–60 μM ethanolamine, and 10–20 mg / L ascorbic acid was dissolved in 500 ml Stempro-34 SFM complete medium at room temperature and added. GlutaMAX and Primosin were added and mixed to prepare a basal medium.

[0245]

[0246] 4-1-2. Manufacturing working badges

[0247] For wild-type iNK cells (WT-iNK), working medium was prepared by adding antioxidant cocktail, IL2 10 IU to 1000 IU, and IL15 5 ng / ml to 50 ng / ml to the basal medium.

[0248] For genetically engineered iNK cells (upCAR-iNK), the working medium was prepared as follows.

[0249] Day 0: Working medium was prepared by adding antioxidant cocktail, IL2 10 IU~1000 IU, IL15 5 ng / ml~50 ng / ml, IL18 50 ng / ml~250 ng / ml, and IL21 1 ng / ml~20 ng / ml to the basal medium.

[0250] Day5, Day7, Day14: When adding badges, the working badge was added using the same badge as in the case of WT-iNK.

[0251]

[0252] 4-2. Cell harvesting, freezing, thawing, and expansion after differentiation into natural killer cells is complete.

[0253] Common Day 50: After differentiation was completed, cells that had completed differentiation were harvested using the method of Example 2 for various analyses. The medium in the flask was harvested and placed in a 50 ml tube, centrifuged at 300 g for 5 minutes, and differentiation media 4-2 was added. Cells were counted using an NC-200 cell counter to confirm the number of cells for each analysis.

[0254] After confirming the number of harvested iNK cells or upCAR-iNK cells, wash them once with DPBS and add 5x10 6 / ml-1x10 7 / ml concentration, and slowly frozen in cell banker or CS10 freezing medium. Then, the vials containing the cells were placed in a Nalgen tank and frozen overnight (o / n) in a deep freeze, and then immediately stored in liquid nitrogen.

[0255] Frozen vials stored in liquid nitrogen for approximately 1 month or more were removed and rapidly thawed in a 37°C water bath. 1 mL of the thawed cells were mixed with 9 mL of working medium, and centrifuged at 300 g for 5 minutes. The supernatant was removed, 1 mL of new working medium was added, and the cell number was measured.

[0256] To refine the degree of stimulation by genetically engineered feeder cells on Day 0, cultured K562 cells were treated with mitomycin C (MMC) at a concentration of 10 μg / mL to 20 μg / mL and incubated for 40 minutes to 2 hours. Then, the MMC-treated genetically engineered feeder K562 cells were harvested and washed three times with DPBS.

[0257] MMC-treated genetically engineered feeder cells and thawed iNK cells were mixed in an appropriate ratio, placed in working medium, and cultured at 37°C and 5% CO₂ conditions. The initial iNK cell density was 2.5x10 5 cells / ml~1x10 6 Adjust to cells / ml and up to 1x10 6 It was set not to exceed cells / ml.

[0258] Culture was performed for 5 days, and on Day 5, cells were harvested from some wells and counted, and the cell concentration was 2.5x10 5 cells / ml~1x10 6 New working medium was added to ensure a cell / ml range. At this time, the doses of IL-2 and IL-15 were added based on the total culture volume.

[0259] On Day 7, the experiment was conducted in the same manner as the harvesting step of the K562 cells of Day 0 by treating them with MMC, and co-culture was performed by adding MMC-treated genetically modified feeder K562 cells.

[0260] On day 10, culture was performed for 5 days, cells were harvested from some wells, and counted, and the cell concentration was 2.5x10 5 cells / ml~1x10 6 New working medium was added to ensure a cell / ml range. At this time, the doses of IL-2 and IL-15 were added based on the total culture volume.

[0261] On Day 14, cells cultured for 14 days were harvested and FACS analysis was performed (analysis was performed at Days 0, 7, 10, and 14).

[0262]

[0263] [Experimental Example]

[0264] Experimental Example 1. Confirmation of mb4-1BBL and mbIL-21 expression in K562 cells.

[0265] 1-1. Confirmation of mb4-1BBL and mbIL-21 expression according to culture period

[0266] The expression of mb4-1BBL and mbIL-21 according to the culture period was confirmed through flow cytometry (FACS) in K562 feeder cells (aAPC-K562) into which dual plasmids expressing mb4-1BBL and mbIL-21 were introduced using the method of Example 3 above.

[0267] As a result, as shown in Fig. 4a, it was confirmed that the expression of mb4-1BBL and mbIL-21 was maintained for up to 60 days after culture in K562 feeder cells introduced with the mb4-1BBL and mbIL-21 dual plasmids.

[0268]

[0269] 1-2. Confirmation of mb4-1BBL and mbIL-21 expression following MMC treatment

[0270] The expression of mb4-1BBL and mbIL-21 before and after MMC treatment was confirmed through flow cytometry in K562 feeder cells (aAPC-K562) into which a dual plasmid expressing mb4-1BBL and mbIL-21 was introduced using the method of Example 3 above.

[0271] As a result, as shown in Figures 4b and 4c, it was confirmed that the expression of mb4-1BBL and mbIL-21 was maintained without decrease after treatment with MMC, which inhibits the proliferation of feeder cells.

[0272]

[0273] Experimental Example 2. Confirmation of cell proliferation according to the ratio of feeder cells and iNK cells during iNK cell expansion.

[0274] 2-1. Confirmation of morphological changes according to the ratio of feeder cells and iNK cells

[0275] As a result of confirming the morphological changes according to the ratio of K562 feeder cells and iNK cells, into which dual plasmids expressing mb4-1BBL and mbIL-21 were introduced during the expansion of iNK cells using the method of Example 4 above, it was confirmed that a high cell proliferation rate was exhibited when the ratio of feeder cells (aAPC):iNK was 2:1, as shown in Fig. 5a (scale bar = 100 μm).

[0276]

[0277] 2-2. Confirming cell counts according to the ratio of feeder cells and iNK cells

[0278] When the number of proliferated cells was confirmed according to the ratio of K562 feeder cells and iNK cells, into which dual plasmids expressing mb4-1BBL and mbIL-21 were introduced during the expansion of iNK cells using the method of Example 4 above, as shown in Fig. 5b and Table 3 below, it was confirmed that when the ratio of feeder cells (aAPC):iNK was 2:1, a higher cell number and proliferation fold were observed compared to ratios of 0.5:1 and 1:1.

[0279]

[0280]

[0281] 2-3. Confirmation of cell markers according to the ratio of feeder cells and iNK cells

[0282] When expanding iNK cells using the method of Example 4, the expression of markers of iNK cells according to the ratio of K562 feeder cells introducing dual plasmids expressing mb4-1BBL and mbIL-21 and iNK cells was confirmed, as shown in Figures 5c to 5e, the expression of the NK cell marker CD45 was all high, and the T cell marker CD3 was hardly expressed. In addition, when the expression of NK cell-related markers CD16, NKG2A, and NKG2D was confirmed, the expression of the NK cell suppression marker NKG2A was confirmed to decrease as the culture period increased when the ratio of feeder cells (aAPC):iNK was 2:1, and the expression of the activation marker NKG2D was confirmed to be similar.

[0283]

[0284] Experimental Example 3. Confirmation of cell proliferation according to the ratio of feeder cells and iNK cells and cytokine combination during expansion of genetically engineered iNK cells.

[0285] 3-1. Morphological changes according to the ratio of feeder cells and genetically engineered iNK cells and cytokine combinations.

[0286] When genetically engineered iNK cells were expanded using the method of Example 4, morphological changes according to the ratio of genetically engineered iNK cells and cytokine combinations and K562 feeder cells introducing dual plasmids expressing mb4-1BBL and mbIL-21 were confirmed. The case where the feeder cell (aAPC):iNK ratio was 2:1 is shown in Fig. 6a, and the case where it was 3:1 is shown in Fig. 6b (scale bar = 100 μm).

[0287] As a result, it was confirmed that a high cell proliferation rate was observed when the ratio of aAPC:iNK was 3:1 and the cytokines were a combination of IL2, IL15, IL21, and IL18.

[0288]

[0289] 3-2. Confirmation of cell count according to the ratio of feeder cells and genetically engineered iNK cells and cytokine combinations

[0290] When the genetically engineered iNK cells were expanded using the method of Example 4, the number of proliferated cells was confirmed according to the ratio of K562 feeder cells introducing dual plasmids expressing mb4-1BBL and mbIL21 and the genetically engineered iNK cells and the combination of cytokines. As shown in Fig. 6c and Tables 4 (aAPC:iNK=2:1) ​​and 5 (aAPC:iNK=3:1) below, it was confirmed that when the ratio of feeder cells (aAPC):iNK was 3:1 and the cytokines were a combination of IL2, IL15, IL21, and IL18, a high cell number and proliferation multiple were observed.

[0291]

[0292]

[0293] 3-3. Confirmation of cell viability according to the ratio of feeder cells and genetically engineered iNK cells and cytokine combinations.

[0294] When the genetically engineered iNK cells were expanded using the method of Example 4, the ratio of K562 feeder cells introducing dual plasmids expressing mb4-1BBL and mbIL-21 to the genetically engineered iNK cells and the cell viability according to the cytokine combination were confirmed. As shown in Fig. 6d, when the ratio of feeder cells (aAPC):iNK was 2:1 and 3:1, it was confirmed that the cell viability increased when IL18 among the cytokines was present.

[0295]

[0296] 3-4. Identification of cell markers according to the ratio of feeder cells and genetically engineered iNK cells and cytokine combinations.

[0297] The results of confirming the ratio of genetically engineered iNK cells and K562 feeder cells that introduced dual plasmids expressing mb4-1BBL and mbIL-21 and the cell markers (CD45, CD3, CD16, NKG2A, NKG2D, CAR, CD24) according to the cytokine combination during the expansion of genetically engineered iNK cells by the method of Example 4 above are shown in Figures 6e and 6f.

[0298] In addition, the results of flow cytometry analysis to determine the expression of cell markers (CD45, CD3, CD16, NKG2A, NKG2D) according to cytokine combinations when the ratio of aAPC:iNK was 2:1 and 3:1 are shown in Figures 7a to 7d (aAPC:iNK=2:1) ​​and Figures 7e to 7h (aAPC:iNK=3:1).

[0299] As a result, it was confirmed that when the ratio of feeder cells (aAPC):iNK was 3:1 and the cytokines were a combination of IL2, IL15, IL21, and IL18, the expression of NK cell activation markers CD16 and NKG2D increased, and the expression of the inhibitory marker NKG2A decreased.

[0300]

[0301] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0302]

[0303] It is expected that the natural killer cell expansion method according to the present invention can be used to mass-produce natural killer cells differentiated from human induced pluripotent stem cells and easily be used for the development of various immune disease and cancer treatment agents, and thus the present invention has industrial applicability.

Claims

1. A dual plasmid vector containing the membrane bound human 4-1BB ligand (mb4-1BBL) and membrane bound human interleukin-21 (mbIL-21) genes.

2. In paragraph 1, A dual plasmid vector, characterized in that the mb4-1BBL gene comprises a base sequence of sequence number 1 and the mbIL-21 gene comprises a base sequence of sequence number 2.

3. In paragraph 1, The above vector is mEF1α (Mouse elongation factor 1α), Neo r A dual plasmid vector characterized in that it further comprises one or more genes selected from the group consisting of (neomycin resistance gene), and rEF1α (Rat elongation factor 1α).

4. A feeder cell transfected with the dual plasmid vector of any one of claims 1 to 3.

5. In paragraph 4, A feeder cell, characterized in that the feeder cell is a K562 cell.

6. In paragraph 4, A feeder cell characterized in that the feeder cell comprises a cell in which human leukocyte antigen (HLA) expression is inhibited.

7. A method for differentiating stem cells into natural killer cells, comprising a step of culturing separated stem cells in a differentiation medium.

8. In paragraph 7, A method characterized in that the above stem cells are human induced pluripotent stem cells (iPSC).

9. In paragraph 8, A method characterized in that the above human induced pluripotent stem cell is a non-genetically modified human induced pluripotent stem cell or a genetically modified human induced pluripotent stem cell.

10. In paragraph 9, A method characterized in that the genetically engineered human induced pluripotent stem cell is a human induced pluripotent stem cell into which a chimeric antigen receptor (CAR) gene has been introduced.

11. In paragraph 7, A method, characterized in that the differentiation medium comprises at least one medium selected from the group consisting of Essential 8, Essential 6, and Stempro34.

12. In paragraph 11, A method characterized in that the differentiation medium further comprises at least one selected from the group consisting of CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, FMS-like tyrosine kinase 3 ligand (FLT3L), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-15 (IL-15), and SB203580.

13. In paragraph 11 or 12, The differentiation medium is a first medium containing Essential 8, CHIR-99021, BMP4, and VEGF; A second medium containing Essential 6, SB431542, SCF, and VEGF; Medium 3 containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, SCF, and FLT3L; A fourth medium containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, and FLT3L; and A method characterized in that it comprises at least one selected from the group consisting of a fifth medium comprising Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, FLT3L, and SB203580.

14. In paragraph 7, A method characterized in that the above natural killer cells express CD56 and CD45 and do not express CD3.

15. A step of culturing the separated stem cells in a differentiation medium to differentiate them into natural killer cells; and A method for expanding natural killer cells, comprising a step of co-culturing the natural killer cells with the feeder cells of claim 4 in a medium for expanding natural killer cells.

16. In paragraph 15, A method characterized in that the above stem cells are human induced pluripotent stem cells (iPSC).

17. In paragraph 16, A method characterized in that the above human induced pluripotent stem cell is a non-genetically modified human induced pluripotent stem cell or a genetically modified human induced pluripotent stem cell.

18. In paragraph 17, A method characterized in that the genetically engineered human induced pluripotent stem cell is a human induced pluripotent stem cell into which a chimeric antigen receptor (CAR) gene has been introduced.

19. In paragraph 15, A method, characterized in that the differentiation medium comprises at least one medium selected from the group consisting of Essential 8, Essential 6, and Stempro34.

20. In paragraph 19, A method characterized in that the differentiation medium further comprises at least one selected from the group consisting of CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, FMS-like tyrosine kinase 3 ligand (FLT3L), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-15 (IL-15), and SB203580.

21. In paragraph 15, The differentiation medium is a first medium containing Essential 8, CHIR-99021, BMP4, and VEGF; A second medium containing Essential 6, SB431542, SCF, and VEGF; Medium 3 containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, SCF, and FLT3L; A fourth medium containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, and FLT3L; and A method characterized in that it comprises at least one selected from the group consisting of a fifth medium comprising Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, FLT3L, and SB203580.

22. In paragraph 15, The above natural killer cell expansion medium is Stempro34, GlutaMAX, and Primosin; and A method characterized in that it comprises at least one selected from the group consisting of an antioxidant cocktail, interleukin-2 (IL-2), interleukin-15 (IL-15), interleukin-18 (IL-18), and interleukin-21 (IL-21).

23. In paragraph 15, If the above natural killer cells are differentiated from non-genetically modified human induced pluripotent stem cells, the medium for natural killer cell expansion contains Stempro34, Glutamax, Primosin, antioxidant cocktail, IL-2, and IL-15. A method characterized in that, when the natural killer cells are differentiated from genetically engineered human induced pluripotent stem cells, the medium for natural killer cell expansion comprises Stempro34, Glutamax, Primosin, an antioxidant cocktail, IL-2, IL-15, and IL-18, or comprises Stempro34, Glutamax, Primosin, an antioxidant cocktail, IL-2, IL-15, IL-18, and IL-21.

24. In paragraph 23, When the above natural killer cells are differentiated from non-genetically modified human induced pluripotent stem cells, the medium for natural killer cell expansion contains IL-2 at 10 to 1000 IU and IL-15 at 5 to 50 ng / ml, A method characterized in that, when the natural killer cells are differentiated from genetically engineered human induced pluripotent stem cells, the medium for natural killer cell expansion contains IL-2 at 10 to 1000 IU, IL-15 at 5 to 50 ng / ml, IL-18 at 50 to 250 ng / ml, and IL-21 at 1 to 20 ng / ml.

25. In paragraph 15, A method characterized in that the step of culturing in the above natural killer cell expansion medium is performed within 7 to 28 days.

26. In paragraph 15, A method characterized in that the cell number ratio of the feeder cells and natural killer cells is 0.5 to 5:

1.

27. A composition for expanding natural killer cells, comprising the feeder cells of clause 4 as an effective ingredient.

28. A kit for natural killer cell expansion, comprising the feeder cells of Article 4, a differentiation medium, and a natural killer cell expansion medium as active ingredients.

29. In paragraph 28, A kit characterized in that the differentiation medium comprises at least one medium selected from the group consisting of Essential 8, Essential 6, and Stempro34.

30. In paragraph 29, A kit characterized in that the differentiation medium further comprises at least one selected from the group consisting of CHIR-99021, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), SB431542, stem cell factor (SCF), β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, FMS-like tyrosine kinase 3 ligand (FLT3L), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-15 (IL-15), and SB203580.

31. In paragraph 28, The differentiation medium is a first medium containing Essential 8, CHIR-99021, BMP4, and VEGF; A second medium containing Essential 6, SB431542, SCF, and VEGF; Medium 3 containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, SCF, and FLT3L; A fourth medium containing Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, and FLT3L; and A kit characterized by comprising at least one selected from the group consisting of Stempro34, beta-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, IL-3, SCF, IL-7, IL-15, FLT3L, and a fifth medium comprising SB203580.

32. In paragraph 28, The above natural killer cell expansion medium is Stempro34, GlutaMAX, and Primosin; and A kit characterized in that it comprises at least one selected from the group consisting of an antioxidant cocktail, interleukin-2 (IL-2), interleukin-15 (IL-15), interleukin-18 (IL-18), and interleukin-21 (IL-21).

33. In paragraph 28, If the above natural killer cells are differentiated from non-genetically modified human induced pluripotent stem cells, the medium for natural killer cell expansion contains Stempro34, Glutamax, Primosin, antioxidant cocktail, IL-2, and IL-15. A kit characterized in that, when the natural killer cells are differentiated from genetically engineered human induced pluripotent stem cells, the medium for natural killer cell expansion comprises Stempro34, Glutamax, Primosin, an antioxidant cocktail, IL-2, IL-15, and IL-18, or comprises Stempro34, Glutamax, Primosin, an antioxidant cocktail, IL-2, IL-15, IL-18, and IL-21.

34. In paragraph 33, When the above natural killer cells are differentiated from non-genetically modified human induced pluripotent stem cells, the medium for natural killer cell expansion contains IL-2 at 10 to 1000 IU and IL-15 at 5 to 50 ng / ml, A kit characterized in that, when the natural killer cells are differentiated from genetically engineered human induced pluripotent stem cells, the medium for natural killer cell expansion contains IL-2 at 10 to 1000 IU, IL-15 at 5 to 50 ng / ml, IL-18 at 50 to 250 ng / ml, and IL-21 at 1 to 20 ng / ml.

35. Use of a composition containing the feeder cell of Article 4 as an effective ingredient for expanding natural killer cells.

36. Use for the manufacture of a preparation for the expansion of natural killer cells comprising the feeder cells of paragraph 4 as an active ingredient.

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