Method for producing NK cells

Abstract

This invention provides a method for producing NK cells from undifferentiated stem cells without using feeder cells in serum-free culture medium. [Solution] A method for producing NK cells without using feeder cells, comprising culturing undifferentiated stem cells in a serum-free medium containing BMP4 and a GSK-3 inhibitor to form embryoid bodies, culturing them in a serum-free medium containing VEGF, SCF, and a TGF-β inhibitor, culturing them in a serum-free medium without a TGF-β inhibitor containing VEGF and SCF to differentiate them into hematopoietic progenitor cells, culturing them in a serum-free medium containing IL-7, SCF, Flt-3L, IL-15, and IL-3, culturing them in a serum-free medium without an IL-3 containing IL-7, SCF, Flt-3L, and IL-15 to differentiate them into NK progenitor cells, and culturing them in a serum-free medium containing IL-2, IL-7, SCF, Flt-3L, IL-15, and a serum substitute to differentiate them into mature NK cells.

Description

[Technical Field]

[0001] The present invention relates to a method for producing NK (natural killer) cells from undifferentiated stem cells without using feeder cells in serum-free culture medium. [Background technology]

[0002] NK cells are cytotoxic lymphocytes involved in the innate immune system. For example, NK cells can respond to virus-infected cells, tumors, and other pathogens. Therefore, in recent years, their use as active ingredients in therapies that utilize immunodeficiency and immune responses has been expanding.

[0003] For use as a pharmacokinetic component in pharmaceuticals, it is preferable to use NK cells that have been differentiated and expanded (proliferated) from undifferentiated stem cells, considering the stability and safety of their supply. Generally, feeder cells are used in the expansion culture of NK cells. For example, Patent Document 1 discloses a method for producing natural killer cells from undifferentiated stem cells, in which undifferentiated stem cells are aggregated by centrifugation in serum-free medium to form centrifugal embryoid bodies, these centrifugal embryoid bodies are cultured in serum-free medium containing BMP4 (Bone Morphogenetic Protein 4), vascular endothelial growth factor (VEGF), and stem cell factor (SCF) to differentiate them into hematopoietic progenitor cells, the obtained hematopoietic progenitor cells are cultured in serum-free medium containing IL-7 (Interleukin-7), SCF, Flt-3L (FMS-like tyrosine kinase 3 ligand), IL-15 (Interleukin-15), and IL-3 (Interleukin-3) to produce NK cells, and further, the NK cells are co-cultured with inactivated artificial antigen-presenting cells (aAPC) in serum-free medium containing IL-2 (Interleukin-2) to expand the culture. Furthermore, in clinical applications, NK cells used are human chronic myeloid leukemia-derived K562 cells that have been irradiated.

[0004] On the other hand, methods using feeder cells are not considered clinically safe, and there is a need for the development of feeder-free methods for expanding NK cell culture. One example of a feeder-free method for expanding NK cell culture is disclosed in Patent Document 2, which describes a method in which human pluripotent stem cells are cultured in a serum-free medium containing BMP4 to obtain mesodermal cells, these mesodermal cells are cultured and differentiated in a serum-free medium containing BMP4, bFGF, and a GSK-3 inhibitor, but not a TGF-β receptor inhibitor, and then cultured and expanded in a serum-free medium containing one or more selected from the group consisting of SCF, Flt-3L, IL-3, IL-7, and IL-15, but not VEGF, bFGF, BMP4, and a Rho kinase (ROCK) inhibitor. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2013 / 163171 [Patent Document 2] Patent No. 6836803 [Overview of the project] [Problems that the invention aims to solve]

[0006] When culturing NK cells in a feeder-free environment, there is a problem in that it is difficult to obtain a sufficient quantity of mature NK cells. The present invention aims to provide a method for producing NK cells from undifferentiated stem cells without using feeder cells in serum-free culture medium. [Means for solving the problem]

[0007] The inventors of the present invention have found that by employing a culture process in which cells are cultured in a serum-free medium containing a specific combination of components at each stage of differentiation from undifferentiated stem cells, NK cells can be produced from undifferentiated stem cells without the use of feeder cells. Furthermore, they have discovered that the NK cells produced by this culture process have high cytotoxicity, are sufficiently mature, and also have good proliferative capacity, thus completing the present invention.

[0008] In other words, the present invention is as follows: [1] A method for producing NK cells from undifferentiated stem cells, (1) A step of culturing undifferentiated stem cells in serum-free medium containing BMP4 and GSK-3 inhibitors to form embryoid bodies, (2) A step in which the embryoid bodies obtained in step (1) are cultured in a serum-free medium containing vascular endothelial growth factor, stem cell factor, and a TGF-β inhibitor, and then cultured in a serum-free medium containing vascular endothelial growth factor and stem cell factor but not containing a TGF-β inhibitor to differentiate them into hematopoietic progenitor cells, (3) The cells obtained in step (2) above are cultured in a serum-free medium containing IL-7, stem cell factor, Flt-3L, IL-15, and IL-3, and then cultured in a serum-free medium containing IL-7, stem cell factor, Flt-3L, and IL-15 but not IL-3 to differentiate them into NK progenitor cells. (4) A step of differentiating the cells obtained in step (3) into NK mature cells by culturing them in a serum-free medium containing IL-2, IL-7, stem cell factors, Flt-3L, IL-15, and a serum substitute, A method for producing NK cells, which has the following characteristics and performs cell culture throughout the entire process without using feeder cells. [2] The method for producing NK cells according to [1], wherein the culture time in step (1) is 18 to 30 hours. [3] A method for producing NK cells according to [1] or [2], wherein step (2) is performed by culturing for 42 to 54 hours in a serum-free medium containing vascular endothelial growth factor, stem cell factor, and a TGF-β inhibitor, and then culturing for 18 to 78 hours in a serum-free medium containing vascular endothelial growth factor and stem cell factor but not containing a TGF-β inhibitor. [4] A method for producing NK cells according to any of [1] to [3] above, wherein step (3) is performed by culturing for 162 to 174 hours in a serum-free medium containing IL-7, stem cell factor, Flt-3L, IL-15, and IL-3, and then culturing for 18 to 510 hours in a serum-free medium containing IL-7, stem cell factor, Flt-3L, and IL-15, but not containing IL-3. [5] A method for producing NK cells according to any of [1] to [4], wherein step (4) is performed by culturing the cells for 234 hours or more in a serum-free medium containing IL-2, IL-7, stem cell factor, Flt-3L, IL-15, and a serum substitute. [6] A method for producing NK cells according to any of [1] to [5] above, wherein the GSK-3 inhibitor is CHIR99021. [7] A method for producing NK cells according to any of [1] to [6] above, wherein the TGF-β inhibitor is SB431542. [8] A method for producing NK cells according to any of [1] to [7], wherein the culture in step (1) is carried out in a culture vessel with low cell adhesion. [9] A method for producing NK cells according to any of the above [1] to [8], wherein NK cells expressing one or more selected from the group consisting of KIR, CD16, NKG2D, NKp44, and NKp46 are produced.

[10] A method for producing NK cells having cytotoxic properties, according to any of the above [1] to [9].

[11] A method for producing NK cells according to any of the above [1] to

[10] , wherein the undifferentiated stem cells are iPS cells. [Effects of the Invention]

[0009] By the method for producing NK cells according to this embodiment, NK cells can be produced from undifferentiated stem cells without using feeder cells in a serum-free medium. Therefore, the method for producing NK cells according to this embodiment is suitable for producing NK cells as a raw material for pharmaceuticals that require high safety.

Brief Description of Drawings

[0010] [Figure 1] FIG. 1(A) is a diagram schematically showing the culture process of Example 1, and FIG. 1(B) is a diagram schematically showing the culture process of Comparative Example 1. [Figure 2] FIG. 2(A) is a diagram showing the results of surface antigen analysis of embryoid bodies on the 6th day of culture in Example 1, and FIG. 2(B) is a diagram showing the results of surface antigen analysis of embryoid bodies on the 6th day of culture in Comparative Example 1. [Figure 3] FIG. 3 is a diagram showing the results of flow cytometry of CD56+CD45+ cells obtained in (4) of Example 1. [Figure 4] FIG. 4 is a diagram showing the results of flow cytometry of CD56+CD45+ cells obtained in (5) of Example 1. [Figure 5] FIG. 5 is a diagram showing the results of measuring the cell number over time in (5) of Example 1. [Figure 6] FIG. 6 is a diagram showing the results of examining the cytotoxicity of NK cells against tumor target cells when the NK cells obtained in (5) of Example 1 and tumor target cells (K562 cells) were co-cultured at each ratio.

Modes for Carrying Out the Invention

[0011] Hereinafter, the method for producing NK cells of this embodiment will be described in detail.

[0012] In the present invention and this specification, "X1 to X2 (X1 and X2 are real numbers satisfying X1 < X2)" means "X1 or more and X2 or less".

[0013] In the present invention and this specification, "Y +"Cells (where Y is a cell surface antigen)" refers to "cells that express Y (Y-positive cells)."

[0014] The method for producing NK cells according to this embodiment is a method for producing NK cells from undifferentiated stem cells, comprising the following steps (1) to (4), wherein cell culture in all steps is carried out without using feeder cells. (1) A step of culturing undifferentiated stem cells in serum-free medium containing BMP4 and GSK-3 inhibitors to form embryoid bodies. (2) A step in which the embryoid bodies obtained in step (1) are cultured in a serum-free medium containing VEGF, SCF, and a TGF-β inhibitor, and then cultured in a serum-free medium containing VEGF and SCF but not containing a TGF-β inhibitor to differentiate them into hematopoietic progenitor cells. (3) A step in which the cells obtained in step (2) are cultured in a serum-free medium containing IL-7, SCF, Flt-3L, IL-15, and IL-3, and then cultured in a serum-free medium containing IL-7, SCF, Flt-3L, and IL-15 but not IL-3, to differentiate them into NK progenitor cells. (4) A step of differentiating the cells obtained in step (3) into NK mature cells by culturing them in a serum-free medium containing IL-2, IL-7, SCF, Flt-3L, IL-15, and a serum substitute.

[0015] In this embodiment, the method for producing NK cells involves performing cell culture in a serum-free medium throughout the entire process. This serum-free medium is a medium that does not contain serum and is a basal medium containing the minimum nutrients necessary for cell survival, to which various additives are added as needed. Examples of basal media include IMDM (Iscove's Modified Dulbecco's Medium), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimal Essential Medium), MEM (Minimum Essential Medium), MEMα, Opti-MEM, RPMI-16, and modified versions thereof.

[0016] The serum-free culture medium used in this embodiment may contain various components commonly found in mammalian cell culture media, to the extent that it does not impair the effects of the present invention. Examples of such components include insulin, transferrin (apo), sodium selenite, ethanolamine, amino acids, vitamins, etc. The culture medium may also contain antibiotics such as penicillin and streptomycin.

[0017] In the present invention and this specification, "serum substitute" refers to any component that can be used in place of serum in cell culture, and is not particularly limited.

[0018] In the present invention and this specification, "NK cells" refers to CD45 + CD56 + These are the cells. In this embodiment, NK cells include NK progenitor cells and NK mature cells. NK progenitor cells express NKp46 but do not express KIR, CD16, NKG2D, and NKp44. On the other hand, NK mature cells express NKp46 in addition to at least one of KIR, CD16, NKG2D, and NKp44.

[0019] In this embodiment, the NK cells may be NK cells into which a chimeric antigen receptor (CAR) has been introduced.

[0020] In the method for producing NK cells according to this embodiment, the culture conditions other than the culture medium composition and culture time are acceptable as long as they allow the stem cells used and the target NK cells to be cultured. For example, when using human undifferentiated stem cells, they can be cultured at 37°C in a 5% CO2 environment. Furthermore, the cell density is appropriately adjusted according to the type of cells used so as not to excessively reduce the cell's proliferative capacity.

[0021] In the present invention and this specification, "undifferentiated stem cells" refer to undifferentiated cells that have the ability to self-replicate and possess pluripotency (the ability to differentiate into various cell types). The undifferentiated stem cells used as raw materials in this embodiment may be any undifferentiated stem cells that can ultimately differentiate into NK cells, and are not particularly limited. Examples of such undifferentiated stem cells include pluripotent stem cells such as ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells), as well as somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, and skin stem cells. In this embodiment, ES cells, iPS cells, hematopoietic stem cells, or neural stem cells are preferred as undifferentiated stem cells, with iPS cells being more preferred.

[0022] Among the undifferentiated stem cells used in this embodiment, ES cells and somatic stem cells can be obtained from animal tissues. For example, hematopoietic stem cells can be collected from bone marrow, umbilical cord, placenta, and peripheral blood. Among the undifferentiated stem cells used in this embodiment, iPS cells can be obtained by dedifferentiating differentiated cells of animals.

[0023] The undifferentiated stem cells used in this embodiment may be those that have been pre-cultured under proliferation conditions that allow for expansion while maintaining their undifferentiated state. These proliferation conditions can be appropriately selected from known conditions and modified as necessary, taking into consideration the type of undifferentiated stem cells used. For example, various differentiation-inducing factor-free media commercially available for culturing undifferentiated stem cells can be used as the culture medium, and these differentiation-inducing factor-free media can also be appropriately modified to use.

[0024] The undifferentiated stem cells used in this embodiment may be cells derived from any species. Mammalian cells are preferred as the undifferentiated stem cells used in this embodiment, more preferably undifferentiated stem cells from humans, monkeys, mice, rats, rabbits, guinea pigs, cattle, horses, pigs, sheep, dogs, cats, etc., even more preferably human undifferentiated stem cells, even more preferably human iPS cells, human mesenchymal stem cells, human hematopoietic stem cells, or human neural stem cells, and particularly preferred human iPS cells.

[0025] The undifferentiated stem cells used in this embodiment may be cells that have been previously introduced with CAR. In this embodiment, CAR-introduced human iPS cells are preferred as the undifferentiated stem cells used.

[0026] The BMP4 used in this embodiment may be a naturally derived protein extracted and purified from the tissues or cells of any organism, or it may be a recombinant protein. It may also be a recombinant protein having the same amino acid sequence as the naturally derived protein, or it may be a recombinant protein that has been modified by introducing mutations that do not impair the function of the naturally derived protein. Furthermore, commercially available BMP4 can also be used in this embodiment.

[0027] The GSK-3 inhibitor used in this embodiment is not particularly limited and can be appropriately selected from known GSK-3 inhibitors. Furthermore, one type of GSK-3 inhibitor may be used, or two or more types of GSK-3 inhibitors may be used. Examples of GSK-3 inhibitors include kaempawornone, 1-azakempawornone, CHIR99021, CHIR98014, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418, lithium, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazole-3-yl)-(2-phenylquinazoline-4-yl)amine, pyridocarbazole-cyclopentadienyl ruthenium complex, and TDZD-8. 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, alpha-4-dibromocetophenone, AR-AO144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridine-3-yl]-4-pyrazine-2-ylpyrrole-2,5-dione, TWS1 Examples include pyrrolopyrimidine compounds, L803H-KEAPPAPPQSpP-NH2 or its myristoylated form, 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-etanone, GF109203X, RO318220, TDZD-8, TIBPO, and OTDZT, with CHIR99021 being preferred. Anti-GSK-3 antibodies may also be used.

[0028] The VEGF used in this embodiment may be derived from any species. In this embodiment, it is preferable that the VEGF used is derived from the same species as the undifferentiated stem cells used. Similarly, the SCF used in this embodiment may be derived from any species, but it is preferable that the SCF is derived from the same species as the undifferentiated stem cells used.

[0029] The VEGF and SCF used in this embodiment may be naturally derived proteins extracted and purified from the tissues or cells of any organism, or they may be recombinant proteins. They may also be recombinant proteins having the same amino acid sequence as the naturally derived protein, or they may be recombinant proteins that have been modified by introducing mutations that do not impair the function of the naturally derived protein. Furthermore, commercially available VEGF and SCF can also be used in this embodiment.

[0030] The TGF-β inhibitor used in this embodiment is not particularly limited, and can be appropriately selected from known TGF-β inhibitors. Furthermore, one type of TGF-β inhibitor may be used, or two or more types of TGF-β inhibitors may be used. Examples of TGF-β inhibitors include SB431542, A83-01, SU5416, SB505124, GC1008, ID11, AP12009, AP11014, LY550410, LY580276, LY364947, LY2109761, SB505124, SB431542, SD208, SM16, NPC30345, Ki26894, SB203580, SD093, Gleevec, 3,5,7,2',4'-pentahydroxyflavone (Morin), activin-M108A, P144, soluble TBR2-Fc, etc., with SB431542 being preferred. Alternatively, an anti-TGF-β receptor antibody may also be used.

[0031] The IL-7, IL-15, IL-3, and IL-2 used in this embodiment may be naturally derived proteins extracted and purified from the tissues or cells of any organism, or they may be recombinant proteins. They may also be recombinant proteins having the same amino acid sequence as the naturally derived proteins, or they may be recombinant proteins that have been modified by introducing mutations that do not impair the function of the naturally derived proteins. Furthermore, commercially available IL-7, IL-15, IL-3, and IL-2 used in this embodiment may also be used.

[0032] Flt-3L is a ligand for the FLT3 receptor, a cell surface receptor (also known as CD135) known as FMS-associated tyrosine kinase 3. The Flt-3L used in this embodiment is not particularly limited and can be any substance capable of transmitting signals into the cell by binding to the FLT3 receptor. For example, it may be the full-length protein of Flt-3L, or a partial peptide of Flt-3L or a mutant peptide thereof that has the ability to bind to the FLT3 receptor.

[0033] The IL-7, SCF, Flt-3L, IL-15, IL-3, and IL-2 used in this embodiment may be derived from any species. Preferably, the IL-7, SCF, Flt-3L, IL-15, IL-3, and IL-2 used in this embodiment are proteins or partial proteins thereof derived from the same species as the undifferentiated stem cells used, or variants thereof.

[0034] In the manufacturing method according to this embodiment, first, as step (1), undifferentiated stem cells are cultured in a serum-free medium containing BMP4 and a GSK-3 inhibitor to form embryoid bodies.

[0035] The BMP4 concentration of the serum-free medium used in the culture in step (1) is not particularly limited as long as it is a concentration capable of forming embryoid bodies from undifferentiated stem cells. For example, the BMP4 concentration of the serum-free medium can be 5 to 500 ng / mL, preferably 10 to 250 ng / mL, more preferably 25 to 125 ng / mL, and even more preferably 25 to 75 ng / mL.

[0036] The concentration of the GSK-3 inhibitor in the serum-free medium used for culture in step (1) is not particularly limited as long as it is a concentration capable of causing undifferentiated stem cells to form embryoid bodies. For example, the concentration of the GSK-3 inhibitor in the serum-free medium can be 0.1 to 250 μM, preferably 1 to 250 μM, more preferably 1 to 50 μM, and even more preferably 1 to 10 μM.

[0037] In the cell culture in step (1), it is preferable to culture undifferentiated stem cells in a culture vessel with low cell adhesion because the formation efficiency of embryoid bodies is good. Examples of culture vessels with low cell adhesion include those whose surface is coated with a material that has low cell adhesion, such as hydrogel.

[0038] The culture time for cell culture in step (1) is not particularly limited, as long as it is sufficient for embryoid bodies to form from undifferentiated stem cells. When using human undifferentiated stem cells, the culture time in step (1) can be, for example, 18 to 30 hours, preferably 20 to 28 hours, and more preferably 24 to 26 hours.

[0039] Next, in step (2), the embryoid bodies obtained in step (1) are cultured in serum-free medium containing VEGF, SCF, and a TGF-β inhibitor, and then cultured in serum-free medium containing VEGF and SCF but not containing a TGF-β inhibitor to differentiate them into hematopoietic progenitor cells. Since hematopoietic progenitor cells express the cell surface antigen CD34, differentiation into hematopoietic progenitor cells can be confirmed using an immune response with an anti-CD34 antibody.

[0040] The VEGF concentration of the serum-free medium used in the culture in step (2) is not particularly limited as long as it is a concentration capable of differentiating embryoid bodies into hematopoietic progenitor cells. For example, the VEGF concentration of the serum-free medium can be 5 to 500 ng / mL, preferably 10 to 250 ng / mL, more preferably 25 to 125 ng / mL, and even more preferably 25 to 75 ng / mL.

[0041] The SCF concentration of the serum-free medium used in the culture in step (2) is not particularly limited as long as it is a concentration capable of differentiating embryoid bodies into hematopoietic progenitor cells. For example, the SCF concentration of the serum-free medium can be 5 to 500 ng / mL, preferably 10 to 250 ng / mL, more preferably 25 to 125 ng / mL, and even more preferably 25 to 75 ng / mL.

[0042] The concentration of the TGF-β inhibitor in the serum-free medium used in the culture in step (2) is not particularly limited as long as it is a concentration capable of differentiating embryoid bodies into hematopoietic progenitor cells. For example, the concentration of the TGF-β inhibitor in the serum-free medium can be 1 to 100 μM, preferably 2 to 50 μM, more preferably 5 to 25 μM, and even more preferably 5 to 15 μM.

[0043] The culture time for cell culture in step (2) is not particularly limited, as long as it is sufficient for the embryoid body to differentiate into hematopoietic progenitor cells. When using human undifferentiated stem cells, the culture time in step (2) can be, for example, cultured in a serum-free medium containing VEGF, SCF, and a TGF-β inhibitor for preferably 42 to 54 hours, more preferably 44 to 52 hours, and then cultured in a serum-free medium containing VEGF and SCF but not containing a TGF-β inhibitor for preferably 18 to 78 hours, more preferably 42 to 78 hours.

[0044] Next, in step (3), the cells obtained in step (2) are cultured in a serum-free medium containing IL-7, SCF, Flt-3L, IL-15, and IL-3, and then cultured in a serum-free medium containing IL-7, SCF, Flt-3L, and IL-15 but not IL-3 to differentiate them into NK progenitor cells. Since NK progenitor cells express the cell surface antigens CD56 and CD45, differentiation into NK progenitor cells can be confirmed by using an immune response with antibodies against these antigens.

[0045] The concentrations of IL-7, SCF, Flt-3L, IL-15, and IL-3 in the serum-free medium used in the culture in step (3) are not particularly limited, as long as they are at concentrations capable of differentiating hematopoietic progenitor cells into NK progenitor cells. For example, the concentrations of IL-7, SCF, Flt-3L, IL-15, and IL-3 in the serum-free medium can each be independently 1 to 500 ng / mL, preferably 2 to 250 ng / mL, more preferably 5 to 100 ng / mL, and even more preferably 5 to 50 ng / mL. When using human undifferentiated stem cells, for example, they can be cultured in serum-free medium containing 20 ng / mL IL-7, 20 ng / mL SCF, 10 ng / mL Flt-3L, 10 ng / mL IL-15, and 5 ng / mL IL-3, and then cultured again by completely changing the medium to serum-free medium containing 20 ng / mL IL-7, 20 ng / mL SCF, 10 ng / mL Flt-3L, and 10 ng / mL IL-15.

[0046] The culture time for cell culture in step (3) is not particularly limited, as long as it is sufficient for hematopoietic progenitor cells to differentiate into NK progenitor cells. When using human undifferentiated stem cells, the culture time in step (3) can be, for example, cultured for 162 to 174 hours, more preferably 166 to 172 hours, in a serum-free medium containing IL-7, SCF, Flt-3L, IL-15, and IL-3, and then cultured for 18 to 510 hours, more preferably 234 to 486 hours, in a serum-free medium containing IL-7, SCF, Flt-3L, and IL-15, but not IL-3.

[0047] Next, in step (4), the cells obtained in step (3) are cultured in a serum-free medium containing IL-2, IL-7, SCF, Flt-3L, IL-15, and a serum substitute to differentiate them into mature NK cells. Since mature NK cells express one or more proteins selected from the group consisting of KIR, CD16, NKG2D, and NKp44, differentiation into mature NK cells can be confirmed by using an immune response with antibodies against these proteins.

[0048] The concentrations of IL-2, IL-7, SCF, Flt-3L, and IL-15 in the serum-free medium used for culture in step (4) are not particularly limited, as long as they are at concentrations that can differentiate NK progenitor cells into mature NK cells. For example, the concentrations of IL-2, IL-7, SCF, Flt-3L, and IL-15 in the serum-free medium can each be independently 1 to 500 ng / mL, preferably 2 to 250 ng / mL, more preferably 5 to 100 ng / mL, and even more preferably 5 to 50 ng / mL.

[0049] The concentration of the serum substitute in the serum-free medium used in the culture in step (4) is not particularly limited as long as it is a concentration capable of differentiating NK progenitor cells into mature NK cells. For example, the concentration of the serum substitute in the serum-free medium is preferably 0.1 to 10% (v / v), and more preferably 1 to 10% (v / v).

[0050] For example, human undifferentiated stem cells can be differentiated into mature NK cells by culturing them in a serum-free medium containing 50 ng / mL IL-2, 20 ng / mL IL-7, 20 ng / mL SCF, 10 ng / mL Flt-3L, 10 ng / mL IL-15, and 5% (v / v) serum substitute.

[0051] The culture time for cell culture in step (4) is not particularly limited, as long as it is sufficient for NK progenitor cells to differentiate into mature NK cells. When using human undifferentiated stem cells, the culture time in step (4) can be, for example, 234 hours or more, more preferably 330 to 342 hours, in a serum-free medium containing IL-2, IL-7, SCF, Flt-3L, IL-15, and a serum substitute.

[0052] In step (4), mature NK cells exhibit increased expression of components involved in cytotoxicity, such as KIR, CD16, NKG2D, NKp44, and NKp46, and thus possess cytotoxic properties. In step (4), CD56 is present in the cell population. +By continuing the culture until the cell ratio exceeds 90%, a cell population can be obtained as a culture that has a high proportion of cells expressing one or more proteins selected from the group consisting of KIR, CD16, NKG2D, and NKp44, and a high proportion of mature NK cells.

[0053] In the NK cell production method according to this embodiment, highly proliferative NK cells can be produced without using serum and feeder cells through the culture process of steps (1) to (4). Furthermore, the proportion of mature NK cells in the resulting cell population is also high. For this reason, the culture product containing NK cells obtained by the NK cell production method according to this embodiment is suitable as an active ingredient in pharmaceutical compositions for cancer immunotherapy, infectious disease treatment, immunodeficiency treatment, and the like.

[0054] Although embodiments of the present invention have been described in detail above, the configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications to the configurations are possible without departing from the spirit of the present invention. [Examples]

[0055] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0056] <Flow Cytometry> Hereafter, unless otherwise specified, cell surface antigens were analyzed using a BD Accuri C6 Flow Cytometer or a BD FACSCanto II (Beckton Dickinson), and the data were analyzed using FlowJo (Beckton Dickinson).

[0057] The antibodies used are as follows: Anti-CD34 antibody: Clone: ​​581 (manufactured by BioLegend) Anti-CD43 antibody: Clone: ​​CD43-10G7 (manufactured by BioLegend) Anti-CD45 antibody: Clone: ​​H130 (manufactured by BioLegend) Anti-CD56 antibody: Clone:5.1H11 (manufactured by BioLegend) Anti-KIR antibody: Clone: ​​DX27 (manufactured by BioLegend) Anti-CD16 antibody: Clone:3G8 (manufactured by BioLegend) Anti-NKG2D antibody: Clone:1D11 (manufactured by BioLegend) Anti-NKp44 antibody: Clone: ​​P44-8 (manufactured by BioLegend) Anti-NKp46 antibody: Clone:9E2 (manufactured by BioLegend)

[0058] [Example 1] iPS cells were differentiated into NK cells in serum-free medium without the use of feeder cells. A schematic diagram of the culture process is shown in Figure 1(A).

[0059] (1) Culture of iPS cells As iPS cells, we used human iPS cell line 253G1 (obtained from the Center for iPS Cell Research and Application, Kyoto University). For culturing iPS cells, we used a 6-well polystyrene tissue culture plate with a surface coated with iMatrix-511 (manufactured by Nippi Corporation). StemFit cell culture medium (manufactured by Ajinomoto Healthy Supply Co., Ltd.) was used as a differentiation-inducing factor-free culture medium.

[0060] 20,000 iPS cells per well were seeded into a 6-well tissue culture plate and cultured overnight in differentiation-inducing factor-free medium in the presence of 10 μM Y-27632 Rock inhibitor (Fujifilm Wako Pure Chemical Industries, Ltd.). The following day, the medium was completely changed to differentiation-inducing factor-free medium to remove the Rock inhibitor. Subsequently, the iPS cells in the tissue culture plate were cultured for 5 days, with the medium being changed as needed to accommodate cell proliferation. After culture, the iPS cells were treated with the cell detachment solution TrypLE Select (Invitrogen, Inc.) for 4-5 minutes and collected as a single-cell dispersion.

[0061] (2) Formation of iPS cell-derived embryoid bodies The single-cell dispersion collected in (1) above was seeded onto a 96-well round-bottom plate (Corning) with an ultra-low adhesion surface (ULA) coated with hydrogel, so that there were 3000 iPS cells per well. Embryoid bodies (EBs) were formed by culturing in serum-free medium containing 50 ng / mL BMP4 and 5 μM CHIR99021 (GSK-3 inhibitor) for 1 day.

[0062] (3) Differentiation of embryoid bodies into hematopoietic progenitor cells The embryoid bodies formed in (2) above were completely replaced with serum-free medium containing 50 ng / mL VEGF, 50 mg / mL SCF, and 10 μM SB431542 (TGF-β inhibitor), and then cultured for 2 days in an incubator at 5% CO2 and 37°C. Subsequently, the medium was replaced with serum-free medium containing 50 ng / mL VEGF and 50 ng / mL SCF, and the cells were cultured for 2 days in an incubator at 5% CO2 and 37°C to obtain hematopoietic progenitor cells.

[0063] (4) Differentiation induction from hematopoietic progenitor cells to NK progenitor cells The hematopoietic progenitor cells obtained in (3) above were seeded in a gelatin-coated polystyrene 6-well tissue culture plate, with 15 cells per well, and cultured for 7 days in serum-free medium containing 20 ng / mL IL-7, 20 ng / mL SCF, 10 ng / mL Flt-3L, 10 ng / mL IL-15, and 5 ng / mL IL-3. Subsequently, the culture medium was completely changed to serum-free medium containing 20 ng / mL IL-7, 20 ng / mL SCF, 10 ng / mL Flt-3L, and 10 ng / mL IL-15, and the culture was continued. After that, half of the medium was changed every 3-4 days for 4-5 times to obtain NK progenitor cells.

[0064] (5) Differentiation induction from NK progenitor cells to mature NK cells The NK progenitor cells obtained in (4) above were cultured in a serum-free medium containing 50 ng / mL IL-2, 20 ng / mL IL-7, 20 ng / mL SCF, 10 ng / mL Flt-3L, 10 ng / mL IL-15, and 5% (v / v) serum substitute, and the culture was continued. Thereafter, half of the medium was changed every 3-4 days to differentiate into mature NK cells and further proliferate (expand) them.

[0065] The series of cultures described in (1) to (5) were performed in three independent trials (N=1 to 3).

[0066] [Comparative Example 1] Figure 1(B) shows a schematic diagram of the culture process in which iPS cells are differentiated into NK cells in serum-free medium using feeder cells.

[0067] (1) Culture of iPS cells Human iPS cell line 253G1 was cultured in the same manner as in Example 1 (1) and collected as a single-cell dispersion.

[0068] (2) Formation of iPS cell-derived embryoid bodies APEL medium (STEM CELL TECHNOLOGY) containing 20 ng / mL BMP4 (R&D systems), 40 ng / mL VEGF (Fujifilm Wako Pure Chemical Industries), and 40 ng / mL SCF (Fujifilm Wako Pure Chemical Industries) was dispensed into a 96-well round-bottom plate (Corning) with an ultra-low adhesion surface (ULA) coated with hydrogel. The single-cell dispersion collected in (1) above was then seeded so that there were 3000 iPS cells per well. The plate was then centrifuged at 300G at room temperature for 5 minutes to aggregate the iPS cells and form embryoid bodies (EBs), and then placed calmly in an incubator at 5% CO2 and 37°C. To ensure the formation of centrifuged EBs in the plate, the cells in the plate were not removed and were cultured for at least 6 days to obtain hematopoietic progenitor cells.

[0069] (3) Differentiation induction from hematopoietic progenitor cells to NK progenitor cells The hematopoietic progenitor cells obtained in (2) above were seeded in a gelatin-coated polystyrene 6-well tissue culture plate, with 15 cells (15EB) per well, and cultured for 7 days in serum-free medium containing 20 ng / mL IL-7 (PEPROTECH), 20 ng / mL SCF (Fujifilm Wako Pure Chemical Industries), 10 ng / mL Flt-3L (Fujifilm Wako Pure Chemical Industries), 10 ng / mL IL-15 (PEPROTECH), and 5 ng / mL IL-3 (Fujifilm Wako Pure Chemical Industries). Subsequently, the culture medium was completely changed to serum-free medium containing 20 ng / mL IL-7, 20 ng / mL SCF, 10 ng / mL Flt-3L, and 10 ng / mL IL-15, and the culture was continued. After that, half of the medium was changed every 3-4 days for 4-5 times to obtain NK progenitor cells.

[0070] (4) Differentiation induction from NK progenitor cells to mature NK cells The NK progenitor cells obtained in (3) above are completely replaced with serum-free medium containing 50 ng / mL IL-2 (Miltenyi Biotech), and co-cultured with irradiated K562 cells. Thereafter, half of the medium is replaced every 3-4 days to differentiate into mature NK cells and further proliferate (expand).

[0071] [Test Example 1] Analysis of surface antigens in hematopoietic progenitor cells Embryoid bodies from Example 1 or Comparative Example 1 were collected on day 6 of culture and digested at 37°C with Accumax solution (Innovative Cell Technologies) for 5-15 minutes to obtain single-cell suspensions. Cells in the single-cell suspensions were stained with anti-CD34 antibody, anti-CD43 antibody, and anti-CD45 antibody. Subsequently, the cells were washed twice with PBS (phosphate saline) containing 2% (v / v) FBS (fetal bovine serum) or PBS. The washed cells were analyzed using a flow cytometer.

[0072] Figure 2(A) shows the results of embryoid bodies on day 6 of culture for Example 1, and Figure 2(B) shows the results of embryoid bodies on day 6 of culture for Comparative Example 1. Hematopoietic progenitor cells were CD34. +They are cells. When cultured in the culture process of Example 1, more than 70% of the cells in the embryoid bodies on the 6th day of culture had differentiated into hematopoietic progenitor cells (Figure 2(A)). In contrast, when cultured in the culture process of Comparative Example 1, the proportion of CD34 + cells in the embryoid bodies on the 6th day of culture was less than 10%.

[0073] [Test Example 2] Analysis of surface antigens of NK progenitor cells and NK mature cells Pipetting was performed on NK progenitor cells (cells recovered in (4) of Example 1 or cells recovered in (3) of Comparative Example 1) to obtain a cell suspension. The cells in the cell suspension were stained with anti-CD45 antibody, anti-CD56 antibody, anti-KIR antibody, anti-CD16 antibody, anti-NKG2D antibody, anti-NKp44 antibody, and anti-NKp46 antibody. Then, the cells were washed twice with PBS containing 2% (v / v) FBS or PBS. The washed cells were analyzed with a flow cytometer. NK mature cells (cells recovered in (5) of Example 1) were also stained with antibodies in the same manner and analyzed with a flow cytometer.

[0074] CD56 in the cells obtained in (4) of Example 1 + CD45 + The results of flow cytometry using the surface antigen markers of the cells are shown in Figure 3. In Figure 3, the dotted line indicates the results of the cell population before staining with the antibody, and the solid line indicates the results of the cell population after staining with the antibody. As shown in Figure 3, the CD56 + CD45 + cells obtained in (4) of Example 1 expressed NKp46, but did not express KIR, CD16, NKG2D, and NKp44, and were confirmed to be NK progenitor cells.

[0075] The number of recovered cells per well and the proportion (%) of CD56 + CD45 + cells (NK cells) in the whole cells, and the number of CD56 + CD45 +Table 1 shows the results of measuring the number of cells. As shown in Table 1, culturing using the culture process of Example 1 yielded a larger quantity of NK precursor cells than culturing using the culture process of Comparative Example 1.

[0076] [Table 1]

[0077] Furthermore, CD56 in the cells obtained in Example 1 (5) + CD45 + Figure 4 shows the results of flow cytometry using surface antigen markers of cells (NK cells). In Figure 4, the dotted line shows the results for the cell population before antibody staining, and the solid line shows the results for the cell population after antibody staining. As shown in Figure 4, the culture process of Example 1 yielded a large number of mature NK cells expressing functional molecules such as KIR, CD16, NKG2D, NKp44, and NKp46. It is possible to mature the cells to a level equivalent to Comparative Example 1 without using feeder cells.

[0078] Furthermore, Figure 5 shows the results of measuring the number of cells over time during the culture process in (5) of Example 1. In Figure 5, "Culture days" refers to the number of culture days from the start of differentiation induction to mature NK cells in the culture process in (5). As shown in Figure 5, mature NK cells were successfully proliferated without feeder cells using the culture process in (5) of Example 1. It is possible to proliferate cells to a level equivalent to Comparative Example 1 without using feeder cells.

[0079] [Test Example 3] In vitro cytotoxicity measurement test of NK cells We targeted tumor cells and used NK cells as effectors to investigate the cytotoxicity of NK cells against tumor cells.

[0080] The target tumor cells (K562 cells) were incubated with CellTrace® Far Red Cell Proliferation Kit, for flow cytometry (Invitrogen) at 37°C for 15 minutes and washed once. Then, these tumor cells were co-cultured with NK cells (cells obtained in Example 1 (5)) in IMDM medium (Nacalai Tesque) containing 10% (v / v) FBS (SIGMA) and 2 mM L-glutamine (Invitrogen) to achieve the indicated effector-to-target (E:T) ratio. 3.5 hours after the start of culture, CellEvent® Caspase-3 / 7 Detection Reagents (Invitrogen) were added, and incubation continued for another 30 minutes. Five minutes before the end of culture, SYTOX® AADvanced® Dead Cell Stain Kit (Invitrogen) was added, and the cells were analyzed using a flow cytometer. The cytotoxicity rate (%) was calculated using the following formula.

[0081] [Cytotoxicity rate (%)]=(1-[FITC during co-culture - AAD - [Percentage of cells (%)] / [FITC during target cell culture alone] - AAD - [Percentage of cells (%)] × 100

[0082] Figure 6 shows the results of investigating the cytotoxicity of NK cells against tumor target cells when co-cultured at various effector-to-target (E:T) ratios. The higher the ratio of NK cells to tumor target cells, the higher the cytotoxicity rate, confirming that the mature NK cells obtained in the culture process of Example 1 possess cytotoxic properties. It is possible to achieve a level of cytotoxicity equivalent to Comparative Example 1 without using feeder cells.

[0083] [Test Example 4] Measurement of cytokine production capacity of NK cells iPS-derived NK cells and tumor target cells (K562 cells) were seeded in 96-well tissue culture plates at a density of 30,000 cells per well. Co-culture was performed for 24 hours in IMDM medium (Nacalai Tesque) containing 10% (v / v) FBS (SIGMA) and 2 mM L-glutamine (Invitrogen). After 24 hours, the plates were centrifuged at 300G for 3 minutes. The resulting culture supernatant was used to quantify the production of IFNγ and TNFα, which are involved in the antitumor function of NK cells, by ELISA.

[0084] [Table 2]

[0085] Table 2 shows the results of measuring the production levels of IFNγ and TNFα by NK cells. As shown in Table 2, iPS-derived NK cells co-cultured with tumor target cells for 24 hours produced IFNγ and TNFα, confirming that they possess antitumor cytokine production ability similar to normal NK cells.

Claims

1. A method for producing NK cells from undifferentiated stem cells, (1) A step of culturing undifferentiated stem cells in a serum-free medium containing BMP4 and a GSK-3 inhibitor to form embryoid bodies, (2) A step of differentiating the embryoid bodies obtained in step (1) into hematopoietic progenitor cells by culturing them in a serum-free medium containing vascular endothelial growth factor, stem cell factor, and a TGF-β inhibitor, and then culturing them in a serum-free medium containing vascular endothelial growth factor and stem cell factor but not containing a TGF-β inhibitor, (3) A step of differentiating the cells obtained in step (2) into NK precursor cells by culturing them in a serum-free medium containing IL-7, stem cell factor, Flt-3L, IL-15, and IL-3, and then culturing them in a serum-free medium containing IL-7, stem cell factor, Flt-3L, and IL-15 but not IL-3, (4) A step of differentiating the cells obtained in step (3) into NK mature cells by culturing them in a serum-free medium containing IL-2, IL-7, stem cell factors, Flt-3L, IL-15, and a serum substitute, A method for producing NK cells, comprising the ability to perform cell culture in all steps without using feeder cells.

2. The method for producing NK cells according to claim 1, wherein the culture time in step (1) is 18 to 30 hours.

3. The method for producing NK cells according to claim 1, wherein step (2) is performed by culturing for 42 to 54 hours in a serum-free medium containing vascular endothelial growth factor, stem cell factor, and a TGF-β inhibitor, and then culturing for 18 to 78 hours in a serum-free medium containing vascular endothelial growth factor and stem cell factor, but not containing a TGF-β inhibitor.

4. The method for producing NK cells according to claim 1, wherein step (3) is performed by culturing for 162 to 174 hours in a serum-free medium containing IL-7, stem cell factor, Flt-3L, IL-15, and IL-3, and then culturing for 18 to 510 hours in a serum-free medium containing IL-7, stem cell factor, Flt-3L, and IL-15, but not containing IL-3.

5. The method for producing NK cells according to claim 1, wherein step (4) is carried out by culturing for 234 hours or more in a serum-free medium containing IL-2, IL-7, stem cell factor, Flt-3L, IL-15, and a serum substitute.

6. The method for producing NK cells according to claim 1, wherein the GSK-3 inhibitor is CHIR99021.

7. The method for producing NK cells according to claim 1, wherein the TGF-β inhibitor is SB431542.

8. The method for producing NK cells according to claim 1, wherein the culture in step (1) is carried out in a culture vessel with low cell adhesion.

9. A method for producing NK cells according to claim 1, wherein NK cells expressing one or more selected from the group consisting of KIR, CD16, NKG2D, NKp44, and NKp46 are produced.

10. A method for producing NK cells according to claim 1, wherein NK cells having cytotoxic properties are produced.

11. The method for producing NK cells according to any one of claims 1 to 8, wherein the undifferentiated stem cells are iPS cells.

Images