WO2020180065A1 - Method for preparation of natural killer cell in cord blood monocyte - Google Patents

Abstract

The present invention relates to a novel marker for isolating cells for differentiation into natural killer cells from cord blood. CD117-positve cells isolated from cord blood have a high degree of differentiation and can be effectively used to prepare natural killer cells having improved activity, compared to conventional markers. Thus, the natural killer cells prepared using the present invention are expected to find various applications.

Description

Method for producing natural killer cells from umbilical cord blood mononuclear cells

The present invention relates to a method for producing natural killer cells, and more particularly, to a method for producing natural killer cells with increased surface antigen expression in the body using CD117-positive cells isolated from cord blood.

Among the cells that make up the human immune system, natural killer cells (NK cells) have the ability to kill cancer cells, cytotoxicity to virus-infected cells, and the ability to kill bacteria and fungi. As various effects such as such have been revealed, various cell therapeutics using natural killer cells have been actively developed. T cells with similar functions can attack cancer cells only by recognizing both the major histocompatibility complex (MHC) and tumor antigens expressed in the target cells. It is not easy to prepare T cells having various antigen specificities targeting various carcinomas because they are different depending on the type. In addition, when the T cell receptor is different from the main histocompatibility complex of a cancer patient, it has a limitation that T cells must be produced from the patient's own blood because it can attack normal cells. Therefore, natural killer cells that can selectively attack only cancer cells, even if they are not cells derived from the patient themselves, are cell therapeutics that can be effectively used for various diseases.

However, since these natural killer cells only exist about 5 to 20% of the lymphocytes in the blood, the amount is not only small to be used separately from peripheral blood, and most of the natural killer cells contained in peripheral blood are completely differentiated. Since they are cells, there is a limit to proliferation in the body after transplantation. Therefore, recently, various methods for producing large amounts of natural killer cells by differentiating cells isolated from umbilical cord blood in vitro have been actively researched. A number of these have been developed (Korean Patent Registration No. 10-0902340). However, unlike natural killer cells in peripheral blood, natural killer cells differentiated in this way are immature, and the expression of surface antigens (receptors) that can selectively attack cancer cells such as KIR, CD2, and CD16 is significantly reduced. Therefore, there is a problem in that the treatment efficiency using natural killer cells decreases.

Therefore, if a method for manufacturing natural killer cells with increased expression of surface antigens, such as natural killer cells in peripheral blood from umbilical cord blood, is developed, natural killer cells with increased therapeutic efficiency can be easily manufactured in large quantities and thus cells of various diseases. It is expected to be widely used as a therapeutic agent.

The present invention was devised to solve the problems of the prior art as described above, a method of producing a natural killer cell with increased expression of a surface antigen using CD117-positive cells contained in umbilical cord blood, prepared by the above method It is intended to provide a natural killer cell, the use of the natural killer cell, and the like.

However, the technical task to be achieved by the present invention is not limited to the above-mentioned tasks, and other tasks that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. will be.

The present invention provides a method for separating cells for differentiation of natural killer cells, including the step of separating cells positive for CD117 (Cluster of Differentiation 117; SCF receptor (stem cell factor receptor); c-kit) from umbilical cord blood.

In addition, the present invention provides a method for producing natural killer cells using the isolated cells for differentiation of natural killer cells. The manufacturing method is preferably (a) separating CD117 (Cluster of Differentiation 117) positive cells from umbilical cord blood; And (b) treating the isolated cells with cytokines to differentiate into natural killer cells, but is not limited thereto, as long as it is generally differentiated into natural killer cells.

In addition, the present invention provides a natural killer cell prepared by the above method.

In addition, the present invention provides a cell therapy comprising natural killer cells prepared by the above method.

In one embodiment of the present invention, the CD117-positive cells may preferably be CD34 (Cluster of Differentiation 34) negative cells, may be CD56 (Cluster of Differentiation 56) positive cells, or CD34 negative and CD56 positive cells The cells may be, preferably, hematopoietic stem cells, natural killer cell progenitor cells, undifferentiated natural killer cells, and the like, more preferably CD34 positive, CD56 negative, and CD117 positive hematopoietic stem cells. (Hematopoietic progenitor cells); CD34 negative, CD56 positive, and CD117 positive immature natural killer cells; It may be a CD34-negative, CD56-negative, and CD117-positive natural killer cell progenitor cell, but is not limited thereto as long as it is a CD117-positive cell isolated from cord blood.

In another embodiment of the present invention, the natural killer cell is characterized in that the surface antigen (receptor) is increased to increase the activity of the cell, preferably CD2 (Cluster of Differentiation 2), CD16 (Cluster of Differentiation 16). ), and KIR (Killer Cell Immunoglobulin-like Receptors), but may have one or more surface antigens selected from the group consisting of, but not limited thereto, if it is a surface antigen related to the activity of natural killer cells.

In another embodiment of the present invention, the cytokine is IL-3 (Interleukin-3), IL-15 (Interleukin-15), SCF (Stem Cell Factor), FLT3L (Fms-related tyrosine kinase 3 ligand), etc. However, it is not limited thereto as long as it is a known cytokine used for differentiation of natural killer cells. Preferably, the cytokine treatment is (a) IL-3 (Interleukin-3), IL-15 (Interleukin-15), SCF (Stem Cell Factor), and FLT3L (Fms-related tyrosine kinase 3 ligand) treatment. Step to do; (b) treating FLT3L and IL-15; And (c) treating IL-15, wherein IL-3 is treated at a concentration of 2 to 8 ng/mL, and IL-15 is treated at a concentration of 5 to 15 ng/mL, and , The SCF is treated at a concentration of 10 to 30 ng/mL, and the FLT3L can be treated at a concentration of 5 to 15 ng/mL.

In another embodiment of the present invention, the manufacturing method can be cultured with feeder cells, and the culture auxiliary cells are generally treated with mitomycin C or X-ray ) Cells that inhibit division and proliferation through irradiation, and produce various metabolites to help the proliferation of target cells. The kind of the culture aid cell is not limited as long as it is a cell that is generally used, and is preferably a cell that overexpresses human delta-like 4 protein (Delta-Like 4; DDL-4).

In addition, the present invention provides a method for preventing recurrence of cancer, or preventing or treating cancer, comprising the step of administering to an individual the natural killer cells prepared by the above method.

In addition, the present invention provides a use for preventing recurrence of cancer of natural killer cells prepared by the above method, or for preventing or treating cancer.

In one embodiment of the present invention, the cancer may be leukemia, breast cancer, ovarian cancer, brain cancer, melanoma cancer, gastric cancer, liver cancer, colon cancer, lung cancer, etc., but limited to cancer that can be treated using natural killer cells. It doesn't work.

When the CD117 marker according to the present invention is used, cells isolated from umbilical cord blood can be used to increase the differentiation efficiency and differentiation speed into natural killer cells, and differentiated natural killer cells can attack cancer cells. And the expression of surface antigens (receptors) such as CD16 is remarkably high. Therefore, the natural killer cells of the present invention can be produced not only to increase the productivity of natural killer cells, but also to produce natural killer cells with improved therapeutic effects in various diseases by the method for producing natural killer cells provided in the present invention. It is expected that it can be widely applied to the treatment of various diseases such as cancer.

1 is a schematic diagram schematically showing a method of separating CD117 positive cells from umbilical cord blood.

2 is a view showing a result of confirming the composition of CD117-positive cells isolated from umbilical cord blood according to an embodiment of the present invention using a flow cytometer.

3 is a schematic diagram schematically showing a method of differentiating CD117-positive cells into natural killer cells according to an embodiment of the present invention.

4 is a result of confirming the phenotype of CD117-positive cells-derived natural killer cells according to an embodiment of the present invention by flow cytometry, (a) is a result of confirming the phenotype of natural killer cells when cultured together with a culture auxiliary cell , (b) is a diagram showing the result of confirming the phenotype of natural killer cells when cultured alone or together with culture aid cells.

5 is a view showing the result of differentiating CD34-negative, CD56-positive, and CD117-positive cells into natural killer cells according to an embodiment of the present invention, and confirming the degree of differentiation into KIR-positive natural killer cells.

In the present invention, CD117-positive cells are isolated from umbilical cord blood using CD117 as a marker, and the CD117-positive cells are used to differentiate into natural killer cells in the presence of human DLL4 overexpressing culture aid cells. In addition to showing a high degree of differentiation, it was confirmed that the natural killer cells prepared by the method of the present invention had remarkably high expression of surface antigens (receptors) such as KIR, CD2, and CD16 that can attack cancer cells. Therefore, it can be confirmed that the natural killer cells prepared using the markers of the present invention can significantly increase the treatment efficiency of diseases such as cancer compared to the natural killer cells prepared using cells isolated from the existing cord blood. Therefore, it is expected to be effectively used in the treatment of various cancers.

In the present specification, the term "natural killer cells (NK cells)" refers to a broad concept that collectively refers to cytotoxic large granular lymphocytes (CLGL). These natural killer cells mature in the liver or bone marrow and attack and destroy cancer cells and virus-infected cells in the body. As a cell responsible for innate immunity, recently, cancer treatment, that is, hematopoietic It can be used in combination as an immunotherapy during hair cell transplantation, surgery, or chemotherapy, or it can be used for the purpose of preventing recurrence of cancer after treatment. In addition, co-administration of monoclonal antibodies such as Rituximab and cytokines such as IL-2 and IL-15 is also under clinical trials and can be applied for various purposes in the treatment of cancer.

In the present specification, “cell therapy” refers to a drug (US FDA regulation) used for treatment, diagnosis, and prevention of cells and tissues manufactured through isolation, culture, and special manipulation from humans. It refers to a drug used for treatment, diagnosis, and prevention through a series of actions such as proliferating or selecting allogeneic or xenogeneic cells in vitro to restore tissue function, or changing the biological properties of cells in other ways. . Cell therapy is largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention specifically refers to a therapy including natural killer cells differentiated from cells isolated from umbilical cord blood. The cell therapeutic agent of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to natural killer cells. In addition, it may be prepared by including one or more additional pharmaceutically acceptable carriers for administration. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffering agents, preservatives, painlessness, etc. for injections. Agents, solubilizers, isotonic agents, stabilizers, and the like can be mixed and used, and in the case of topical administration, a base agent, excipient, lubricant, preservative, etc. can be used. The formulation of the cell therapeutic agent of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. have. Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.

The route of administration of the cell therapy according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Includes sublingual or rectal.

The cell therapy products of the present invention vary according to a number of factors, including the activity of the cell therapy used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. And can be appropriately selected by a person skilled in the art. For example, it may be administered at a dose of 1×10 ⁶ to 1×10 ⁹ cells/kg, preferably 1×10 ⁷ to 1×10 ⁹ cells/kg. The administration may be administered once or several times a day. In addition, when formulated as a liquid unit formulation such as a solution, suspension, or emulsion, it may be administered to a patient at the cell concentration.

In the present specification, “prevention” refers to any action that suppresses or delays onset of diseases such as cancer by administration of natural killer cells according to the present invention.

In the present specification, "treatment" refers to any action in which symptoms such as cancer are improved or beneficially changed by the administration of natural killer cells according to the present invention.

In the present specification, "individual" refers to a subject to which the natural killer cells of the present invention can be administered, and the subject is not limited.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1: Isolation of CD117 positive cells from cord blood

In order to separate CD117-positive cells (CD117+ cells) from umbilical cord blood, an equal amount of phosphate buffered saline (PBS) was added to umbilical cord blood provided for research, placed on a ficoll solution, and centrifuged at 2,000 rpm for 30 minutes. Later, mononuclear cells were isolated from the soft coat. The isolated mononuclear cells were washed twice with a phosphate buffer solution, and then anti-CD117 microbeads (Miltenyi biotec) were added and incubated at 4° C. for 15 minutes. Then, CD117-positive cells were isolated using the MACS system (Miltenyi biotec), and then cultured together with the culture auxiliary cells. The overall separation method is briefly shown in FIG. 1. And CD34-positive cells used as a control were also isolated and cultured in the same manner.

Example 2: Confirmation of the composition of CD117 positive cells

In order to confirm the composition of CD117-positive cells isolated in the same manner as in Example 1, flow cytometric analysis was performed on umbilical cord blood mononuclear cells. In more detail, cells were treated with anti-CD56-PE (eBioscience), anti-CD34-FITC (eBioscience), anti-NKG2A-eFluor710 (eBioscience), or anti-CD161-APC (eBioscience), which are monoclonal antibodies. After that, unbound antibody was removed using a phosphate buffer solution, and fluorescence was measured using a flow cytometer (FACS BD Canto II). The results are shown in FIG. 2.

As shown in FIG. 2, CD117-positive cells present in cord blood were 73% of CD34-positive hematopoietic stem cells (HSC), 9.4% of CD56-positive natural killer cells (NK cells), and NKG2A positive cells. It was confirmed that it was composed of NK progenitor cells, CD161 positive NK progenitor cells, etc., and it was confirmed that markers of T cells (CD3+), B cells (CD19+), and monocytes (CD14+) were not expressed in these cells.

Example 3: Differentiation from CD117 positive cells to natural killer cells

In order to differentiate CD117-positive cells isolated in the same manner as in Example 1 into natural killer cells, CD117-positive cells were inoculated in DMEM:HAM'S F12 (2:1) medium to which 10% human serum was added. , 5 ng/mL of interleukin-3 (IL-3), 10 ng/mL of Fms-related tyrosine kinase 3 ligand (FLT3L), 20 ng/mL of stem A stem cell factor (SCF) and 10 ng/mL of interleukin-15 (IL-15) were added and incubated for 5 days. And in order to remove the culture auxiliary cells, the floating cells were collected and centrifuged at 800 rpm, and then the precipitated cells were replaced with a new medium to which 10 ng/mL of FLT3L and 10 ng/mL of IL-15 were added. After incubation for one day, the medium was replaced with a new medium to which 10 ng/mL of IL-15 was added, and further cultured for 6 days. As feeder cells, EL08.1D2 cells, a murine embryonic liver stromal cell line, or EL08.1D2 (human delta-like 4; hDDL4) overexpressing the human delta-like 4 protein (hDDL4) are overexpressed. EL-DLL4) cells were used. The overall differentiation method is briefly shown in FIG. 3. And in order to confirm the degree of differentiation into natural killer cells and the degree of function (cancer cytotoxicity) of the differentiated natural killer cells, that is, maturity, flow cytometry was performed in the same manner as in Example 2. Anti-CD2-FITC (eBioscience), anti-CD16-APC (eBioscience), and anti-KIR-PE (eBioscience) were used as antibodies. The results are shown in FIG. 4.

As shown in FIG. 4(a), when differentiating into natural killer cells using CD34-positive cells as a control group, CD16-positive natural killer cells showed 6% differentiation and maturity of KIR-positive natural killer cells. , When differentiating into natural killer cells using CD117-positive cells, it was confirmed that the differentiation and maturity of CD16-positive natural killer cells increased to 22% and KIR-positive natural killer cells 20%. In addition, when EL-DDL4 cells were used as culture aids, 60% of CD16-positive natural killer cells and 51% of KIR-positive natural killer cells were found to significantly increase differentiation and maturity. In the case of CD2-positive natural killer cells, when differentiated from CD34-positive cells, it was only 1.7%, but when differentiated from CD117-positive cells, the differentiation and maturity increased by more than 17 times to 29.5%.

In addition, as shown in Fig. 4(b), as a result of confirming the degree of differentiation into natural killer cells in the case of using and not using the culture auxiliary cells, when differentiating from CD34 positive cells, the degree of differentiation into natural killer cells is about 30. %, but when differentiated from CD117-positive cells, the degree of differentiation was approximately 80%, confirming that the differentiation efficiency was increased by 2.5 times or more. As a result of confirming the phenotype of differentiated natural killer cells, it was confirmed that the degree of differentiation of CD2-positive natural killer cells and CD16-positive natural killer cells was significantly higher than that of CD34-positive natural killer cells.

Through the above results, in the case of CD117-positive cells isolated using CD117 as a separation marker, rather than CD34-positive cells, which are previously used separation markers, the efficiency of differentiation into natural killer cells is significantly increased, and CD2 of differentiated natural killer cells , CD16, KIR, etc. were also found to be highly expressed, and it was confirmed that natural killer cells can be produced with high efficiency without the use of culture aids. In addition, in the case of culture aid cells, it was confirmed that the efficiency of differentiation and maturation of natural killer cells can be significantly increased when cells overexpressing human delta-like 4 protein are used compared to conventional cells that are mainly used. .

Example 4: Differentiation from CD34 negative, CD56 positive and CD117 positive cells to natural killer cells

After separating CD117-positive cells in the same manner as in Example 1, CD34-negative, CD56-positive, and CD117-positive cells (CD34-/CD56+/CD117+ cells) were additionally isolated using a flow cytometer (BD FACS LSR II). And it was differentiated into natural killer cells in the same manner as in Example 3, and CD34 positive cells were used as a control. The results are shown in FIG. 5.

As shown in FIG. 5, it was confirmed that the degree of differentiation of KIR-positive natural killer cells was increased by about 4 times when differentiated from CD34-negative, CD56-positive, and CD117-positive cells than when differentiated from CD34-positive cells. .

Through the above results, since the CD117 marker of the present invention can also separate the CD34-CD117+ cell population compared to CD34, which was used as a separation marker for cells for differentiation of natural killer cells, the degree of differentiation into natural killer cells significantly increases. In addition, it was confirmed that the expression of surface antigens (receptors) such as KIR, CD2, and CD16 that can attack cancer cells, that is, the maturity, is remarkably high. Therefore, it was confirmed that if natural killer cells were prepared using the CD117 marker of the present invention, not only the production rate of natural killer cells could be increased, but also the therapeutic effect of diseases such as cancer could be remarkably increased.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains can understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

Since the production method of natural killer cells using the CD117 marker according to the present invention can significantly increase the productivity of natural killer cells with improved therapeutic effects, it can be widely and effectively applied to the treatment of various diseases using natural killer cells.

Claims (16)

1. A method for separating cells for differentiation of natural killer cells, comprising the step of separating CD117 (Cluster of Differentiation 117) positive cells from umbilical cord blood.
2. The method of claim 1,

   The CD117-positive cells are characterized in that including CD34 (Cluster of Differentiation 34) negative cells, separation method.
3. The method of claim 1,

   The CD117-positive cells are characterized in that containing CD56 (Cluster of Differentiation 56) positive cells, separation method.
4. The method of claim 1,

   The natural killer cell is characterized in that it has at least one surface antigen selected from the group consisting of CD2 (Cluster of Differentiation 2), CD16 (Cluster of Differentiation 16), and KIR (Killer Cell Immunoglobulin-like Receptors), separation method .
5. (a) separating CD117 (Cluster of Differentiation 117) positive cells from umbilical cord blood; And

   (b) treating the isolated cells with cytokines to differentiate into natural killer cells.
6. The method of claim 5,

   The CD117-positive cells are characterized in that containing CD34 (Cluster of Differentiation 34) negative cells, the production method.
7. The method of claim 5,

   The CD117-positive cells are characterized in that containing CD56 (Cluster of Differentiation 56) positive cells.
8. The method of claim 5,

   The treatment of the cytokine is

   (a) treating IL-3 (Interleukin-3), IL-15 (Interleukin-15), SCF (Stem Cell Factor), and FLT3L (Fms-related tyrosine kinase 3 ligand);

   (b) treating FLT3L and IL-15; And

   (c) comprising the step of treating IL-15.
9. The method of claim 8,

   The IL-3 is treated at a concentration of 2 to 8 ng/mL,

   The IL-15 is treated at a concentration of 5 to 15 ng/mL,

   The SCF is treated at a concentration of 10 to 30 ng/mL,

   The FLT3L is characterized in that the treatment at a concentration of 5 to 15 ng / mL, manufacturing method.
10. The method of claim 5,

    The manufacturing method is characterized in that cultivation with a feeder cell (feeder cell).
11. The method of claim 10,

    The culture auxiliary cells are human Delta-Like 4 protein (human Delta-Like 4; hDDL-4) characterized in that the overexpressing cells, manufacturing method.
12. The method of claim 5,

    The natural killer cell is characterized in that it has one or more surface antigens selected from the group consisting of CD2 (Cluster of Differentiation 2), CD16 (Cluster of Differentiation 16), and KIR (Killer Cell Immunoglobulin-like Receptors), manufacturing method .
13. A natural killer cell manufactured by the method of claim 5.
14. A cell therapeutic agent comprising, as an active ingredient, natural killer cells prepared by the method of any one of claims 5 to 12.
15. A method for preventing or treating cancer, comprising administering to an individual the natural killer cells prepared by the method of any one of claims 5 to 12.
16. The use of the natural killer cells produced by the method of any one of claims 5 to 12 for the prevention or treatment of cancer.

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