WO2022145832A1 - Induced pluripotent stem cell-derived natural killer cell and use thereof - Google Patents

Abstract

The present invention relates to an induced pluripotent stem cell (iPSC)-derived natural killer cell (NK cell), a use thereof, and a preparation method thereof.

Description

Inversely differentiated stem cell (IPSC)-derived natural killer cells and uses thereof

The present invention relates to iPSC-derived natural killer cells (“NK cells”), uses thereof, and methods for preparing the same.

NK cells are lymphoid cells involved in the immune response. These cells have various functions, and in particular, have the function of killing tumor cells, cells with oncogenic transformation, and other abnormal cells in vivo, and are important components of the innate immune system.

NK cells do not attack normal cells expressing MHC class I molecules, but mainly attack cells with reduced or deficient MHC class I molecule expression. Since the expression of MHC class I molecules is decreased in cancer cells or virus-infected cells, NK cells can attack these cells. Therefore, when allogenic NK cells are used as a cell therapy for cancer and infections, there is no need to immunize NK cells in advance to recognize the target cells, and there is a side effect of graft-versus-host disease (GVHD). has the advantage of avoiding it. When NK cells obtained from fresh peripheral blood mononuclear cells (PBMCs) from an actual cancer patient and a normal donor who are close to the recipient are concentrated and then transplanted, the transplanted NK cells do not cause adverse effects on the recipient. It did not occur and survived for a short time and maintained cell killing activity (see Non-Patent Documents 1 and 2). However, clinical trials showing the effectiveness of NK cell transplantation therapy have not yet been reported.

Recently, instead of obtaining NK cells from a donor, a technique for culturing NK cells in vitro has been developed. In particular, for acquired immunotherapy using NK cells, NK cell populations are effectively and efficiently expanded and cultured, and their functions (killing ability, transport ability, localization, persistence and proliferation) are maintained and strengthened in vivo. There is a need for a more improved method for doing so.

[Prior art literature]

(Non-Patent Document 1) Miller, J. S. et al., Blood, 105: 3051 (2005)

(Non-Patent Document 2) Rubnitz, J. E. et al., J. Clin. Oncol., 28: 955 (2010)

Although NK cells can be clinically used for diseases requiring immune enhancement, there are still difficulties in using them as immune cell therapeutics because it is difficult to culture and proliferate NK cells in vitro.

Accordingly, the present invention intends to provide a method for producing NK cells, specifically, a method for producing NK cells derived from induced pluripotent stem cells (“iPSCs”).

In addition, the present invention is to provide NK cells prepared by the above method and their use as a cell therapy agent.

The present invention provides a method for producing NK cells derived from retrodifferentiated stem cells. The manufacturing method of the present invention may include the following steps.

(a) culturing by seeding the retrodifferentiated stem cells on a plate;

(b) culturing by replacing the cultured cells with a medium to which bone morphogenetic protein 4 (BMP4) is added;

(c) culturing by replacing the cultured cells of step (b) with a medium to which vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are added;

(d) culturing by replacing the cultured cells of step (c) with a medium to which SB431542, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) are added;

(e) interleukin-3 (IL-3), stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine in the cultured cells of step (d) culturing by replacing the medium with the kinase 3 ligand (FLT-3L); and

(f) Stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine kinase 3 ligand (FLT-3L) in the cultured cells of step (e) Culturing by replacing with the added medium.

The manufacturing method of the present invention,

(i) in step (b) BMP4 is added at a concentration of 30 to 70 ng/mL, such as 50 ng/mL,

(ii) in step (c) VEGF and bFGF are each added at a concentration of 30 to 70 ng/mL, such as 50 ng/mL, or

(iii) in step (d) at a concentration of 5-50 μM, such as 20 μM, SB431542, 30-70 ng/mL, such as 50 ng/mL, bFGF, 30-70 ng/mL, such as 50 ng/mL or added to

(iv) in step (e) IL-3, SCF, IL-7, IL-15 and FLT-3L are each added at a concentration of 30 to 70 ng/mL, such as 50 ng/mL, or

(v) In step (f), SCF, IL-7, IL-15 and FLT-3L may each be added at a concentration of 30 to 70 ng/mL, such as 50 ng/mL.

In another embodiment, the manufacturing method of the present invention comprises:

(i) culturing the cells in step (b) for 1 to 5 days, or

(ii) culturing the cells in step (c) for 1 to 5 days, or

(iii) culturing the cells in step (d) for 1 to 5 days, or

(iv) culturing the cells in step (e) for 3 to 10 days, or

(v) in step (f) may be to culture the cells for 10 to 30 days.

In another embodiment, the present invention provides a method for producing NK cells derived from idiopathic stem cells, comprising the following steps.

(e) Interleukin-3 (IL-3), stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine kinase 3 Ligand (FLT-3L) is replaced with the added medium and culturing; and

(f) replacing the cultured cells with a medium supplemented with stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine kinase 3 ligand (FLT-3L) culturing step.

The present invention also provides NK cells prepared according to the above production method, and a pharmaceutical composition for treating or preventing cancer comprising the NK cells.

The method for producing iPSC-derived NK cells according to the present invention can effectively proliferate NK cells from a small amount of iPSCs, and thus can be usefully used for mass production of NK cells.

In addition, the NK cells prepared according to the present invention exhibit excellent cell killing ability, and can be usefully used in various fields of immune cell therapy.

1 shows an embodiment of preparing NK cells from iPSCs according to Example 1.

2 is a photograph taken with an optical microscope of the cells generated in each step of Example 1.

3 and 4 show the cancer cell killing ability of NK cells prepared according to the present invention.

The present invention provides a method for producing natural killer cells derived from immunized stem cells, comprising one or more of the following steps.

(a) culturing by seeding the retrodifferentiated stem cells on a plate;

(b) culturing by replacing the cultured cells with a medium to which bone morphogenetic protein 4 (BMP4) is added;

(c) culturing by replacing the cultured cells of step (b) with a medium to which vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are added;

(d) culturing by replacing the cultured cells of step (c) with a medium to which SB431542, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) are added;

(e) interleukin-3 (IL-3), stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine in the cultured cells of step (d) culturing by replacing the medium with the kinase 3 ligand (FLT-3L); and

(f) Stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine kinase 3 ligand (FLT-3L) in the cultured cells of step (e) Culturing by replacing with the added medium.

As used herein, the term "natural killer cell" or "NK cell" refers to a core innate immune cell that performs the primary defense (innate immunity) function in the body to eliminate infection of viruses, bacteria and parasites and abnormal autologous cells (cancer cells, etc.) to be. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus to enter the circulation. NK cells differ from natural killer T cells (NKTs) in terms of phenotype, origin and respective effector functions, and often NKT cell activity promotes NK cell activity by secreting IFNγ. Unlike NKT cells, NK cells do not express the T-cell antigen receptor (TCR) or the pan T marker CD3 or the surface immunoglobulin (Ig) B cell receptor, but generally do express the surface markers CD16 (FcγRIII) and CD56 in humans. and express NK1.1 or NK1.2 in C57BL/6 mice.

As used herein, the term "immunely differentiated stem cells" is also called "induced Pluripotent Stem Cells" or its abbreviation iPSC in English, and is also called "induced pluripotent stem cells". iPSC refers to cells that have pluripotency, i.e., the ability to differentiate into all cells of our body, like embryonic stem cells, through artificial stimulation of multidifferentiated somatic cells, not stem cells.

Methods for preparing iPSCs may use methods well known in the art. For example, if fibroblasts are cultured and then reprogramming factors, such as four genes, Oct4, Sox2, Klf4, and c-Myc, are transferred to fibroblasts using a retrovirus, they differentiate into embryonic stem cells. It forms a cell mass with ability, which is called iPSC. The difference of iPSC from embryonic stem cells and somatic cell cloned embryonic stem cells, which are known as treatable cells, is that there is no ethical problem because embryos are not sacrificed to produce these cells. that it doesn't.

As used herein, the term “culture” may refer to one or more cells that either undergo or do not undergo cell division in an in vitro environment. The in vitro environment can be any medium known in the art suitable for maintaining cells in vitro, such as, for example, a suitable liquid medium or agar.

In step (a), iPSCs may be cultured or maintained in an undifferentiated state using various methods. In one embodiment, iPSCs can be maintained and cultured in an undifferentiated state using a feeder-independent culture system, such as TeSR medium. As used herein, the term "feeder (cell)" refers to a cell that cannot divide and proliferate, but has metabolic activity, and thus helps the proliferation of a target cell by producing various metabolites.

TeSR medium includes, but is not limited to, mTeSR1, TeSR2, or TeSR-E8. Specific information on the medium can be found, for example, on the homepage of Stemcell Technologies, or its usage can be confirmed through known prior literature.

A preferred feeder-free medium for step (a) may be a serum-free medium, such as mTeSR1. Complete mTeSR™1 medium (Basal Medium + 5X Supplement) contains recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ), no other growth factors are required.

A variety of substrate components can be used to culture or maintain iPSCs. For example, collagen IV, fibronectin, laminin, and vintronectin can be used to provide a solid support for iPSC culture and maintenance. In one embodiment, a Matrigel® matrix such as Corning® Matrigel® hESC-Qualified Matrix (Corning Catalog #354277) can be used. Matrigel® is a mixture of gelatinous proteins secreted by mouse tumor cells, the mixture mimics the complex extracellular environment found in many tissues, and serves as a substrate for cell culture. Another example of a substrate may be used with a Vitronectin matrix, such as Vitronectin XF™ (Catalog #07180, a matrix developed and manufactured by Nucleus Biologics).

The medium of step (a) may further contain a ROCK inhibitor.

In a specific example, in step (a), iPSCs may be cultured in Matrigel-coated wells containing mTeSR1 and ROCK inhibitor, and at this time, may be seeded at 5 x 10 ⁴ / well.

In one embodiment, iPSCs can be cultured or differentiated into other cells, such as hematopoietic progenitor cells or hematopoietic stem cells.

As used herein, the term “differentiation” refers to the process by which a less specialized cell can become a more specialized cell in culture or in vivo than it would have under the same conditions without it being differentiated. Under certain conditions, the ratio of differentiated cells (ie, specialized cells) to undifferentiated cells (ie, less specialized cells) may be at least about 1%, 5%, 25% or more.

According to various differentiation methods of iPSCs, hematopoietic stem cells, immune effector cells (eg T cells, NK cells, iNKT cells), myocytes (eg cardiomyocytes), neurons, beta islet cells, kidney cells, lung cells, iPSCs can be differentiated into cell lineages comprising fibroblasts, epidermal cells, or tissues or organs derived therefrom.

Hematopoietic stem cells can be generated using conventionally known culture conditions and using feeder cells. For example, after partial, essentially, or complete isolation or individualization of iPSCs, the cells can be further cultured in a defined medium to promote hematopoietic differentiation. Certain combinations of growth factors can substantially promote differentiation of iPSC cells into hematopoietic stem cells or hematopoietic cell lineages. In certain embodiments, the sequential use of specific combinations of growth factors is important for the hematopoietic differentiation of iPSCs. For example, a combination of one or more of bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and SB431542 may be used.

In a specific embodiment, iPSCs are cultured in a medium comprising BMP4, followed by culture in a medium comprising VEGF and bFGF, and then cultured in a medium comprising SB431542, VEGF and bFGF to another cell, such as hematopoietic stem cells or hematopoietic cells. It can differentiate into progenitor cells.

As a medium for differentiation of iPSCs, a differentiation medium known in the art may be used. In one embodiment, a serum-free and animal component-free basic medium such as STEMdiff™ APEL™ medium (Stem Cell Technologies) can be used. In particular, when specific cytokines are added to STEMdiff™ APEL™ medium, direct differentiation into hematopoietic cells is possible. For a specific protocol for this, it can be found on the homepage of Stem Cell Technologies, or its usage can be confirmed through known prior literature.

In one embodiment, the method for producing NK cells of the present invention may include one or more of the following steps (b) to (d), for example, all of the steps (b) to (d). In the steps (b) to (d) of the present invention, the iPSC-cultured cells may be differentiated into other cells, such as hematopoietic stem cells or hematopoietic progenitor cells.

(b) culturing cells cultured from iPSCs by replacing them with a medium to which bone morphogenetic protein 4 (BMP4) is added;

(c) culturing by replacing the cultured cells of step (b) with a medium to which vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are added; and

(d) culturing by replacing the cultured cells of step (c) with a medium supplemented with SB431542, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).

The cells cultured from the iPSCs in step (b) may be cells cultured in step (a).

In one embodiment, (i) in step (b) BMP4 is added at a concentration of 30 to 70 ng/mL, preferably 40 to 60 ng/mL, more preferably 50 ng/mL;

(ii) VEGF and bFGF are each added in step (c) at a concentration of 30 to 70 ng/mL, preferably 40 to 60 ng/mL, more preferably 50 ng/mL,

(iii) in step (d) SB431542 is 5 to 50 μM, preferably 10 to 40 μM, more preferably 20 μM, VEGF is 30 to 70 ng/mL, preferably 40 to 60 ng/mL, more Preferably 50 ng/mL, bFGF may be added at a concentration of 30 to 70 ng/mL, preferably 40 to 60 ng/mL, more preferably 50 ng/mL.

Each of the cells in steps (b) to (d) may be cultured for 1 to 10 days, preferably 1 to 5 days, such as 2 to 3 days. For example, in step (b), it may be cultured for 3 days, in step (c), it may be cultured for 2 days, and in step (d), it may be cultured for 2 days.

In one embodiment, cells cultured or differentiated from iPSCs are capable of differentiating into NK cells. At this time, the medium for differentiation into NK cells may include various cytokines.

As used herein, the term “cytokine” refers to an immune activating cytokine that can be used to induce cells cultured or differentiated from iPSCs into NK cells. The cytokine is, for example, IL (Interleukin)-3, IL-15, IL-21, FLT3-L (FMS-like tyrosine kinase 3 ligand), SCF (Stem Cell Factor), IL-7, IL-18, It may be IL-4, type I interferons, Granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or Insulin-like growth factor 1 (IGF 1), but is not limited thereto. In one embodiment, a combination of IL-3, SCF, IL-7, IL-15 and FLT-3L or a combination of SCF, IL-7, IL-15 and FLT-3L may be used.

The cytokine is added to the differentiation medium at a concentration of 30 to 70, preferably 40 to 60, more preferably 50 ng/mL.

In one embodiment, the manufacturing method of the present invention may include the following steps (e) and/or (f), preferably steps (e) and (f). Through steps (e) and (f) of the present invention, iPSC-cultured cells (eg, hematopoietic stem cells) can be differentiated into NK cells.

(e) replacing the cells cultured from the iPSC with a medium supplemented with IL-3, SCF, IL-7, IL-15 and FLT-3L and culturing; and

(f) culturing by replacing the cultured cells with a medium to which SCF, IL-7, IL-15 and FLT-3L are added.

The cells cultured from the iPSCs in step (e) may be cells cultured through one or more of steps (b) to (d), such as hematopoietic stem cells.

In one embodiment, in step (e) IL-3, SCF, IL-7, IL-15 and FLT-3L are 30 to 70 ng/mL, preferably 40 to 60 ng/mL, more preferably 50 added at a concentration of ng/mL, or

In step (f) SCF, IL-7, IL-15 and FLT-3L are each added at a concentration of 30 to 70 ng/mL, preferably 40 to 60 ng/mL, more preferably 50 ng/mL. can

The cells in step (e) may be cultured for 3 to 10 days, preferably 4 to 7 days, such as 6 days. The cells in step (f) may be cultured for 10 to 30 days, preferably 15 to 20 days, such as 18 days. At this time, the medium of step (f), such as the NK differentiation medium containing SCF, IL-7, IL-15 and FLT-3L, can be regularly replaced, and a fresh medium is replaced every 6 days when cultured for a total of 18 days. Can be replaced. For example, in step (e), the culture may be performed for 6 days, and in step (f), it may be cultured for 18 days, and the medium may be replaced every 6 days.

The NK differentiation medium may be, for example, Matrigel-coated medium, and includes conventionally known substances such as DMEM, HAM's F12, Glutamax, Human serum, Penicillin Streptomycin, L-glutamine, Beta-mercaptoethanol, Ethanolamine, Ascorbic acid, Sodium selenite, etc. However, it is not limited thereto.

NK cells prepared according to the production method of the present invention exhibit the protein expression characteristics of conventional NK cells. In one embodiment, the NK cell is capable of expressing one or more proteins selected from the group consisting of CD45, CD56, CD2, CD16, CD94, KIR2D, and CD158. In another embodiment, the NK cells may not express one or more proteins selected from the group consisting of CD3, CD19 and CD14. For example, CD56(+) NK cells may be cells expressing cell surface CD56 glycoprotein, or cells expressing CD56 glycoprotein and not expressing CD3 glycoprotein, if necessary. In this case, the CD56(+) NK cells are, for example, CD56+ NK cells in which nonspecific binding is removed by adding CD56 microbeads to PBMC, or specific binding by adding CD3 microbeads to PBMC. After removal of (specific binding), non-specific binding may be removed by adding CD56 microbeads to CD3(-)/CD56(+) NK cells.

In one embodiment, the NK cells produced according to the method of the present invention may be CD45(+)CD56(+) NK cells, or CD3(-)CD19(-)CD14(-) NK cells. In another example, the NK cells prepared according to the method of the present invention are CD3(-)CD19(-)CD14(-) and CD45(+)CD56(+) cells, i.e., CD45(+)CD56 (+)/CD3(-)CD19(-)CD14(-) NK cells.

In another embodiment, the NK cells produced according to the methods of the present invention exhibit, in addition to the characteristics of said NK cell markers, markers directly related to the function of NK cells. For example, the NK cells prepared according to the method of the present invention may express one or more proteins selected from the group consisting of CD2, CD16, CD94, KIR2D, and CD158, that is, CD2(+) NK cells, CD16(+) NK cells, CD94(+) NK cells, or KIR2D, CD158(+) NK cells. In another example, the NK cells produced according to the method of the present invention are CD2(+)/CD56(+) NK cells, CD16(+)/CD56(+) NK cells, CD94(+)/CD56(+) NK cells. cells, or KIR2D, CD158(+)/CD56(+) NK cells.

The NK cells produced according to the present invention may be about 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more of the final prepared cells.

The present invention also provides NK cells prepared according to the above production method.

The NK cells prepared above can be used for cell therapy, in particular for treating or preventing cancer. Accordingly, the present invention provides a pharmaceutical composition for treating or preventing cancer comprising NK cells.

As used herein, the term "cell therapy agent" refers to the biological characteristics of cells by proliferating and selecting living autologous, allogenic, and xenogenic cells in vitro or by other methods to restore the functions of cells and tissues. It refers to medicines used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing the The NK cells of the present invention can be classified as immune cell therapeutics for regulating immune responses, such as suppression of immune responses or enhancement of immune responses in vivo.

The administration route of the anticancer cell therapy composition of the present invention may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, may be administered intradermally, but is not limited thereto.

The composition of the present invention may be formulated in a suitable form together with a pharmaceutically acceptable carrier generally used for cell therapy. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid, sucrose, albumin, and the like. Suitable preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, and polyvinylpyrrolidone.

In another embodiment of the present invention, there is provided a method for preventing or treating cancer comprising administering to a subject an anticancer cell therapy composition comprising NK cells prepared according to the production method of the present invention. The subject means a mammal, preferably a human.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1: Preparation of Induced Pluripotent Stem Cells (iPSC)-derived Natural Killer Cells (iPSC-derived Natural Killer Cells: iPSC-NK)

mTeSR-1 medium was added to a 24-well plate coated with Matrigel, and iPSCs were seeded at a density of 5 x 10 ⁴ cells per well. The seeded cells were differentiated while replacing the medium by time as shown in FIG. 1 . Specifically, on the first day after seeding, the medium was replaced with STEMdiff APEL 2 (Stem cell Technologies), and BMP4 (50 ng/mL, Peprotech) was added to the medium. On day 4 after seeding, VEGF (50 ng/mL, Peprotech) and bFGF (50 ng/mL, Peprotech) were added and the medium was replaced with fresh STEMdiff APEL 2. Six days after seeding, SB431542 (20 μM, Reagents Direct, Encinitas, CA, USA), VEGF (50 ng/mL) and bFGF (50 ng/mL) were added, and the medium was replaced with fresh STEMdiff APEL. On day 8 after seeding, IL-3 (50 ng/mL, Peprotech), SCF (50 ng/mL, Peprotech), IL-7 (50 ng/mL, Peprotech), IL-15 (50 ng/mL, Peprotech) , FLT-3L (50 ng/mL, Peprotech) was added and replaced with fresh NK cell differentiation medium (see Table 1 for composition). On day 14 after seeding, SCF (50 ng/mL, Peprotech), IL-7 (50 ng/mL, Peprotech), IL-15 (50 ng/mL), FLT-3L (50 ng/mL, Peprotech) were added. Then, the medium was replaced with a fresh NK cell differentiation medium, and the above-mentioned cytokines were added and medium was replaced every 6 days until the 32nd day. The generation of differentiated cells at each medium change was observed under an optical microscope. The supernatant was collected and centrifuged at 300 Х g for 10 minutes to obtain cells released into the supernatant.

Composition of NK cell differentiation media

name	manufacturing company	Cat #	Volume	Final Conc.
DMEM (high glucose)	Gibco	11995-065	280 mL	56.60%
HAM's F12	Gibco	11765-054	140 mL	28.30%
Glutamax	Gibco	35050-061	4.2 mL	1X
human serum	Biowest	S4190-100	75 mL	15%
Penicillin Streptomycin	Gibco	15140122	5 mL	One%
L-glutamine	Gibco	25030081	5 mL	2 mM
Beta-mercaptoethanol	Sigma	M3148	0.035 μL	1uM
Ethanolamine	Sigma	E0135	250 μL	50uM
Ascorbic acid	Sigma		A8960	100 μL	20ug/ml
Sodium selenite	Sigma	S5261	2.5 μL	5ng/ml

The results photographed with an optical microscope are shown in FIG. 2 . iPSCs grew in the form of colonies until 1 day after seeding, and then started to change shape after the 4th day. In addition, undifferentiated endothelial spread was seen until day 14, but after day 26, it was confirmed that all of them were differentiated into single-cell type NK cells.

Example 2: Confirmation of NK cell differentiation

In this Example, in order to confirm the characteristics of the cells prepared in Example 1, the expression of NK cell-related markers was confirmed by immunofluorescence staining.

2.1: Confirmation of NK cell differentiation using NK cell markers

For iPSC-derived NK cells differentiated according to Example 1, the expression of CD3(-), CD19(-), CD14(-), CD45(+), CD56(+), which are known NK cell markers, was investigated. did Specifically, after seeding, iPSC-derived NK cells were differentiated up to 8 or 32 days after seeding, and the expression of gastric markers in the differentiated cells was confirmed by flow cytometry. In flow cytometry, a cell surface staining method was used, and more specifically, the cells obtained in Example 1 were subjected to Fixable Viability Dye eFluor™ 506 (live-dead cells) (Invitrogen, Carlsbad , CA, USA), a monoclonal antibody anti- CD3-APC (BD, Franklin Lakes, New Jersey , USA), anti-CD19-APC (BD), anti-CD14-APC-eFluor 780 (Invitrogen), anti-CD45-eFluor 450 (Invitrogen), anti-CD56- Incubated with Alexa Fluor® 488 (BD) antibody. After washing the cells with a buffer, the fluorescence intensity was measured. In addition, the appropriate isotype antibody was stained for comparison. Cell marker expression was analyzed with a flow cytometer (MACSQuant Analyzer 10 Flow Cytometer, Miltenyi biotec, Bergisch Gladbach, Germany).

The above experiment was repeated 3 times, and the experimental results are summarized and shown in Table 3. On the 8th day after differentiation (Day 8), CD3(-)CD19(-)CD14(-) cells (%) were 98.1 ± 1.3%, and CD3(-)CD19(-)CD14(-) intracellular cells (%) ) in CD45(+)CD56(+) (%), it was 9.7 ± 10.2%, whereas on the 32nd day after differentiation (Day 32), it increased to 90.1 ± 8.5% and 89.2 ± 6.9%, respectively, and the total number of cells It increased about 317.2 times.

Day	Total cell number (Fold change)	CD3(-) CD19(-) CD14(-) cell (%)	CD45(+)CD56(+) / CD3(-)CD19(-)CD14(-) cells (%)
Day 0	2.5x10 5	-	-
Day 8	7.52 x10 6 (30.0)	98.1 ± 1.3	9.7 ± 10.2
Day 32	79.3 x10 6 (317.2)	90.1 ± 8.5	89.2 ± 6.9

From this, it was confirmed that the cells differentiated by the method of the present invention were NK cells, and furthermore, it was confirmed that NK cells could be produced from iPSCs with high efficiency according to the method of the present invention (that is, about 90% are NK cells). In particular, it was observed that the NK cell marker increased between Day 8 and Day 32, and cultured in a medium supplemented with IL-3+SCF+IL-7+IL-15+FLT-3L (step e of FIG. 1 ), followed by SCF +IL-7+IL-15+FLT-3L was changed to the added medium and cultured (step f in FIG. 1 ), it was confirmed that the degree of differentiation into NK cells was greatly increased.

2.2: Confirmation of NK cell differentiation using NK cell receptor markers

In Example 2.1, it was confirmed that the already terminally differentiated cells exhibited the characteristics of NK cell markers. In this example, it was attempted to once again confirm the characteristics of terminally differentiated cells through markers directly related to the function of NK cells. Specifically, after differentiation up to 8 days or 32 days after iPSC-derived NK seeding, the expression of markers in the differentiated cells was confirmed by flow cytometry. As a flow cytometry method, the same cell surface staining method as described in Example 2.1 was used, but as a monoclonal antibody, anti-CD56-Alexa Fluor® 488 (BD), anti-CD2-APC (BD), anti-CD16- Cell marker expression was analyzed using APC-H7 (BD) and anti-CD94-FITC (BD) antibodies.

The results are shown in Table 3. Compared to Day 8, CD56(+) increased on Day 32, and when CD2(+), CD16(+), and CD94(+) (%) were analyzed in intracellular (%) of CD56(+), these It was confirmed that all increased.

Day	Total cell number (Fold change)	CD56(+) cells (%)	CD2(+)/ CD56(+) cells (%)	CD16(+)/ CD56(+) cells (%)	CD94(+)/ CD56(+) cells (%)
Day 0	2.5x10 5	-	-	-	-
Day 8	7.52 x10 6 (30.0)	63.0 ± 30.6	0.1 ± 0.0	1.5 ± 1.0	0.5 ± 0.2
Day 32	79.3 x10 6 (317.2)	90.2 ± 7.6	27.4 ± 8.7	17.0 ± 7.6	47.0 ± 7.0

Cells differentiated by the method of the present invention from Examples 2.1 and 2.2 were identified as NK cells, and further, it was confirmed that NK cells could be produced from iPSCs with high efficiency according to the method of the present invention. In particular, it was seen that the expression rate of NK cell-related markers increased between Day 8 and Day 32, and cultured in a medium supplemented with IL-3+SCF+IL-7+IL-15+FLT-3L (step e of FIG. 1) , then it was confirmed that when the culture was changed to the medium added with SCF+IL-7+IL-15+FLT-3L (step f in FIG. 1), the degree of differentiation into NK cells was greatly increased.

Example 3: Confirmation of cancer cell treatment effect of NK cells

In this example, the cancer cell treatment effect of NK cells prepared according to the present invention was evaluated.

3.1: Blood cancer cell preparation (K562: Target cell)

The blood cancer cell line K562 maintained in the T25 cell culture flask was released by pipetting in a 37°C CO ₂ incubator, transferred to a 15 mL conical tube, and centrifuged at 516 x g for 5 minutes. After centrifugation was completed and the supernatant was suctioned, 1 mL of 10% RPMI-1640 medium (Gibco) was added and resuspended. After additional addition of 4 mL of medium and resuspension, the cells were counted. Then, in order to classify the experimental group according to the presence or absence of CFSE staining, the cells were transferred to a 15 mL conical tube as many as the number of cells shown in Table 4, centrifuged at 516 x g for 5 minutes, the supernatant was suctioned, and the medium as much as the volume in Table 4 was added and resuspended.

Group	number of cells	Resuspension Medium Volume
CFSE (X)	1x10 6 cells	2 mL
CFSE (O)	4x10 6 cells	5 mL

After that, the process of adding CFSE was added to the CFSE (O) experimental group. Specifically, 4 µL of 2.5 mM stock CFSE (Invitrogen) was added to the 15 mL conical tube that had been subjected to the previous process so that the final concentration was 2 µM, and then vortexed immediately so that the CFSE staining was uniform. Then, it was put in a 37 ℃ CO ₂ incubator and incubated for 15 minutes, then 7.5 mL of the medium was added, followed by centrifugation at 516 x g for 5 minutes. After centrifugation was completed, the supernatant was suctioned, 10 mL of medium was added, and centrifuged at 516 x g for 5 minutes. After suctioning the supernatant, 1 mL of medium was added and resuspended to release the cells well. 7 mL of medium was additionally added so that the cell density of K562 cells was 5x10 ⁵ /mL.

3.2: NK cell preparation (iPSC-derived NK cells: Effector cell)

The iPSC-derived NK cells differentiated according to the present invention were filtered once in a 70 μm strainer and transferred to a 50 mL conical tube, and the number of cells was counted. Thereafter, as shown in Table 5, serial dilution was performed for each E:T ratio (effector to target cell ratio).

Specifically, 3 5 mL round bottom polystyrene test tubes were prepared for each group, and 100 μL of the NK cells prepared according to Example 1 were added. For tube A (10:1) with the highest E:T ratio, transfer 5x10 ⁶ NK cells to a 15 mL conical tube, centrifuge at 516 x g for 5 minutes, suction the supernatant, add 1 mL of medium, and reproduce It was cloudy. In the case of tube B, tube C, tube D, and tube E, serial dilution was performed using cells from tube A, tube B, tube C, and tube D, respectively.

Tube (E:T ratio)	cell density	Volume of medium	Volume of NK cells
A (10:1)	5x10 6 /mL	-	5x10 6 /mL
B (5:1)	2.5x10 6 /mL	500 μL	500 μL of A dilution
C (2:1)	1x10 6 /mL	600 μL	400 μL of B dilution
D (1:1)	5x10 5 /mL	500 μL	500 μL of C dilution
E (0.5:1)	2.5x10 5 /mL	500 μL	500 μL of D dilution

3.3: Measurement of cancer cell cytotoxicity through co-culture of iPSC-derived NK cells (Effector cells) and blood cancer cells (K562: Target cells)

Prepare a 5 mL round bottom polystyrene test tube, and put 100 μL of K562 cells (target cells) of the CFSE (O) group prepared in step 3.1 each. At this time, in order to measure the spontaneous killing value of the target cell, a separate 5 mL round bottom polystyrene test tube was prepared, and only 100 μL of the target cell was added. The prepared test tube was centrifuged at 516 x g for 1 minute, incubated at 37 ° C. CO ₂ in an incubator for 4 hours, 5 μL of 7-AAD (BD) was added to each tube, and cytotoxicity was measured by flow cytometry.

The results are shown in Table 6, FIGS. 5 and 6 . As the proportion of NK cells increased, the degree of death of the blood cancer cell lines also increased to about 2.8%, about 4.2%, about 12.1%, and about 18.6%.

From this, it was confirmed that the NK cells differentiated by the method of the present invention effectively kill cancer cells, thereby exhibiting a cancer cell therapeutic effect.

Claims (11)

1. A method for producing natural killer cells derived from immunized stem cells, comprising the steps of:

   (a) culturing by seeding the retrodifferentiated stem cells on a plate;

   (b) culturing by replacing the cultured cells with a medium to which bone morphogenetic protein 4 (BMP4) is added;

   (c) culturing by replacing the cultured cells of step (b) with a medium to which vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are added;

   (d) culturing by replacing the cultured cells of step (c) with a medium to which SB431542, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) are added;

   (e) interleukin-3 (IL-3), stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine in the cultured cells of step (d) culturing by replacing the medium with the kinase 3 ligand (FLT-3L); and

   (f) Stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine kinase 3 ligand (FLT-3L) in the cultured cells of step (e) Culturing by replacing with the added medium.
2. The method according to claim 1, wherein in step (a), the retrodifferentiated stem cells are cultured in a feeder-free serum-free medium (feeder-free, serum-free medium).
3. According to claim 1,

   (i) BMP4 is added at a concentration of 30 to 70 ng/mL in step (b), or

   (ii) VEGF and bFGF are each added at a concentration of 30 to 70 ng/mL in step (c), or

   (iii) SB431542 is added at a concentration of 5 to 50 μM, VEGF is 30 to 70 ng/mL, and bFGF is added at a concentration of 30 to 70 ng/mL in step (d);

   (iv) in step (e) IL-3, SCF, IL-7, IL-15 and FLT-3L are each added at a concentration of 30 to 70 ng/mL, or

   (v) in step (f), characterized in that SCF, IL-7, IL-15 and FLT-3L are added at a concentration of 30 to 70 ng/mL, respectively,

   Methods of making natural killer cells.
4. According to claim 1,

   (i) BMP4 is added at a concentration of 50 ng/mL in step (b), or

   (ii) VEGF and bFGF are each added at a concentration of 50 ng/mL in step (c), or

   (iii) SB431542 is added at a concentration of 20 μM, VEGF is 50 ng/mL, and bFGF is 50 ng/mL in step (d);

   (iv) IL-3, SCF, IL-7, IL-15 and FLT-3L are each added at a concentration of 50 ng/mL in step (e), or

   (v) in step (f), characterized in that SCF, IL-7, IL-15 and FLT-3L are each added at a concentration of 50 ng/mL,

   Methods of making natural killer cells.
5. According to claim 1,

   (i) culturing the cells in step (b) for 1 to 5 days, or

   (ii) culturing the cells in step (c) for 1 to 5 days, or

   (iii) culturing the cells in step (d) for 1 to 5 days, or

   (iv) culturing the cells in step (e) for 3 to 10 days, or

   (v) characterized in that the cells in step (f) are cultured for 10 to 30 days,

   Methods of making natural killer cells.
6. The method of claim 1, wherein the natural killer cells express one or more proteins selected from the group consisting of CD45, CD56, CD2, CD16, CD94, KIR2D, and CD158.
7. The method of claim 1, wherein the natural killer cells do not express one or more proteins selected from the group consisting of CD3, CD19 and CD14.
8. A method for producing natural killer cells derived from immunized stem cells, comprising the steps of:

   (e) Interleukin-3 (IL-3), stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine kinase 3 Ligand (FLT-3L) is replaced with the added medium and culturing; and

   (f) replacing the cultured cells with a medium supplemented with stem cell factor (SCF), interleukin-7 (IL-7), interleukin-15 (IL-15) and FMS-like tyrosine kinase 3 ligand (FLT-3L) culturing step.
9. The method of claim 8, wherein the cells cultured from the retrodifferentiated stem cells in step (e) are hematopoietic stem cells.
10. 10. A natural killer cell prepared according to any one of claims 1 to 9.
11. A pharmaceutical composition for treating or preventing cancer, comprising natural killer cells prepared according to any one of claims 1 to 9.

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