KR101447546B1 - A method for differentiation and expansion of NK cell from CD14 positive monocytes - Google Patents

Abstract

More particularly, the present invention relates to a method for proliferating and differentiating natural killer cells (NK cells) from cord blood, and more particularly, to a method for proliferating and differentiating natural killer cells (NK cells) from cord blood, ^- CD34 ^- CD56 ^- CD19 ^- cells; Preparing a CD14 ⁺ cells, 2) the CD3 ^- CD34 ^- CD56 ^- CD19 ^- removing the CD14-negative cells from the cells to ^{CD3 - CD34 - CD56 - - CD19} ; And 3) culturing the CD3 ^- CD34 ^- CD56 ^- CD19 ^- CD14 ⁺ cells after IL-15 and IL-21 are mixed and cultured, thereby efficiently multiplying and differentiating natural killer cells from cord blood . The present invention relates to a method for inducing NK cells from CD14-positive cells in a short period of time, in comparison with a method for inducing NK cells from hematopoietic stem cells, and a method for inducing NK cells having excellent cell- And can be used for the treatment of cancer cells because it can differentiate NK cells having cancer cell kill function.

Description

Technical Field [0001] The present invention relates to a method for differentiation and proliferation of NK cell from CD14 positive monocytes,

The present invention relates to the differentiation and proliferation of natural killer cells (NK cells), and more particularly to a method for efficiently promoting the differentiation and proliferation of CD14-positive cells derived from mononuclear cells of cord blood into NK cells .

Of the cells constituting the immune system, natural killer cells (hereinafter referred to as "NK cells") are known to be capable of killing cancer nonspecifically. These NK cells can be used for the treatment of solid tumors using lymphocine activated killer cells (LAK) and tumor infiltration lymphocytes (TIL), or for the donor lymphocyte Bordignon C, et al., Hematologia 84: 1110-1149, 1999) by performing an immunotherapy with an immunosuppressive agent (Tilden, AB et al., J. Immunol . , 136: 3910-3915, 1986; New cell therapies have been attempted to prevent rejection of organ transplants. In addition, defects in the differentiation and activity of NK cells were observed in breast cancer (Konjevic G, et al., Breast Cancer Res . Treat . , 66: 255-263, 2001), Melanoma (Ryuke Y, et al., Melanoma Res ., 13: 349-356, 2003), lung cancer (Villegas FR , et al. Lung Cancer , 35: 23-28, 2002) have been reported to be related to various cancer diseases, NK cell therapy is emerging to treat these diseases.

B cells and T cells are found in rats deficient in the expression of γ _c of Cytokine receptors but receptors with γ _c play an important role in NK differentiation in that NK cells are not found (Singer , B et al., Proc. Natl. Acad. Sci. USA 92, 377-381, 1995). The receptors γ _c are receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-2 enhances proliferation and activation of mature NK cells (Shibuya, A. et al., Blood 85, 3538-3546, 1995). In humans and mice deficient in IL-2, the number of NK cells is remarkably reduced (DiSanto, JP et al., J. Exp . Med . 171, 1697-1704, 1990) And IL-2Ra deficiency indirectly affect the number and activation of NK cells. In addition, the IL-2R chain is known to be involved in the formation of receptors for IL-15.

IL-15 is involved in NK cell differentiation, which is deficient in NK cells in mice lacking the transcription factor interferon (IFN) -regulatory factor 1 required for IL-15 production (Kouetsu et al., Nature 391, 700-703, 1998), and that NK cells were not found in rats deficient in IL-15 or IL-15Ra. It has been reported that IL-15 directly promotes the growth and differentiation of NK cells through the IL-15 receptor expressed in NK cells (Mrozek et al., Blood 87, 2632-2640, 1996).

IL-21 is a cytokine secreted by activated CD4 ⁺ T cells (Nature, 5: 688-697, 2005), receptors of IL-21 (IL-21R) are dendritic cells, NK cells, Like cells (Rayna Takaki, et al., J. Immonol 175: 2167-2173, 2005). IL-21 is structurally very similar to IL-2 and IL-15, and IL-21R shares a chain with IL-2R, IL-15, IL-7R and IL-4R (Asao et al., J Immunol , 167: 1-5, 2001). IL-21 has been reported to induce the maturation of NK cell precursors from bone marrow (Parrish-Novak, et al., Nature , 408: 57-63, 2000), and in particular the cytokine production and cytotoxicity ( J. Immunol , 170: 5464-5469, 2003; J. Brady, et al., J Immunol , 172: 2048-2058, 2004), and it has been reported that CD8 ⁺ T cells also increase the function of the effector, thereby promoting the anticancer response of the immune and adaptive immune system (Rayna Takaki, et al., J Immunol 175: 2167-2173, 2005; J Immunol , 173: 900-909, 2004). In addition, it activates NK cells isolated from human peripheral blood (Parrish-Novak, et al., Nature , 408: 57, 2000) and plays an important role in inducing mature NK cells from hematopoietic stem cells isolated from cord blood (J. Brady, et al., J Immunol , 172: 2048, 2004).

In order to effectively utilize NK cells for anti-cancer immunotherapy, it is necessary to acquire a large number of NK cells. However, NK cells account for 10-15% of lymphocytes in the blood, and the number, differentiation and function of NK cells are often lowered in cancer patients. Therefore, mass production of NK cells through proliferation and differentiation of NK cells is required for application as an NK cell therapy agent.

NK cells are known to be derived from the hematopoietic stem cells of the bone marrow. In vitro, there have been reported methods of differentiating hematopoietic stem cells from cord blood into NK cells by culturing and culturing appropriate cytokines (Immunity 3: 459-473, 1995; Blood 87: 2632-2640, 1996 Eur J Immunol 33: 3439-3447, 2003, Blood 108: 3824-3833, 2006). In other words, Flt-3L, IL-7, SCF and IL-15 can be added to CD34 ⁺ HSC for 4 weeks to differentiate into CD3 ^- CD56 ⁺ NK cells. However, such a differentiation method has difficulties in practical clinical application, and it is difficult to obtain a sufficient amount of cells for treatment, and there are many difficulties in actual clinical application such as requiring time and cost for differentiation.

Therefore, the present inventors developed CD3 ^- CD34 ^- CD56 ^- CD19 ^- cells by removing CD3, CD34, CD56 and CD19 - positive cells from mononuclear cells isolated from cord blood while developing a method for obtaining NK cells more efficiently and economically then, the CD3 ^- removal of the CD14 negative cells from the cell CD3 ^{- -} CD34 ^- CD56 ^- CD19 was produced CD14 ⁺ cells ^- CD34 ^- CD56 ^- CD19. Then, the method of culturing the CD3 ^- CD34 ^- CD56 ^- CD19 ^- CD14 ⁺ cells after the mixed treatment with IL-15 and IL-21 significantly promotes the differentiation and proliferation of NK cells, NK cells can be induced in a short time, thereby completing the present invention.

It is an object of the present invention to provide a method for promoting the differentiation and proliferation of NK cells having excellent cell killing ability by mixing IL-15 and IL-21 with cord blood-derived CD14-positive mononuclear cells.

In order to achieve the above object, the present invention provides a method of differentiating NK cells from CD14-positive mononuclear cells, comprising the step of mixing IL-15 and IL-21 with cord blood-derived CD14 positive mononuclear cells.

The present invention also provides a method for proliferating NK cells from CD14-positive mononuclear cells, comprising culturing cord blood-derived CD14 positive mononuclear cells with IL-15 and IL-21.

The present invention also provides a pharmaceutical composition for preventing and treating cancer comprising NK cells prepared by the method according to the present invention as an active ingredient.

The method for differentiating and proliferating CD14-positive cells into NK cells according to the present invention is characterized in that CD3, CD34, CD56 and CD19-positive T cells, B cells and NK cells are removed from monocytes isolated from cord blood, And IL-21 can significantly promote the differentiation and proliferation of NK cells, and can induce NK cells having excellent cytotoxic activity in a short time, thereby being useful for the treatment of cancer cells have.

FIG. 1 shows the recovery rate of CD14-positive cells after the removal of CD3-positive cells by removing mononuclear cells (MNC) from human umbilical cord blood and then CD34, CD19 and CD56-positive cells were sequentially removed and CD14- .
FIG. 2 is a graph showing the purity of CD34-positive cells and the purity of CD14-positive cells, respectively, by two-color flow cytometric analysis, and the numbers on the right are the percentages of cells corresponding to each quadrant.
FIG. 3 shows the results of immunohistochemical staining of monocytes isolated from cord blood and removing CD3-positive cells, followed by removal of CD34, CD19 and CD56-positive cells and analysis of CD14-positive cells obtained by FACS using CD15, CD19, CD33, CD117 and CD122 markers And the percentage of cells corresponding to the quadrant.
FIG. 4 is a graph showing the results of differentiation of CD34 positive hematopoietic stem cells and CD14 positive mononuclear cells, which are the source of NK cells from umbilical cord blood, and NK differentiation on the 7th day after culturing on NK differentiation medium containing IL-15 and IL- CD3, CD56, and CD122 markers to identify the percentages of cells in the quadrant identified by FACS.
FIG. 5 is a graph showing the distribution of NK differentiation on day 12 when CD34-positive hematopoietic stem cells and CD14-positive mononuclear cells, which are the source of NK cells from cord blood, were separated and cultured in NK differentiation medium containing IL-15 and IL- CD3, CD56, and CD122 markers to identify the percentages of cells in the quadrant identified by FACS.
FIG. 6 is a diagram showing the fold induction of cell repair by culturing CD34-positive hematopoietic stem cells and CD14-positive mononuclear cells in an NK differentiation medium containing IL-15 and IL-21. FIG.
FIG. 7 is a graph showing the ⁵¹ Cr release assay of NK cells differentiated from CD34-positive hematopoietic stem cells for 28 days and NK cells having 90% or more differentiation for 12 days from CD14-positive mononuclear cells.
E: T ratio is the ratio of effector to target cell.

Hereinafter, the present invention will be described in detail.

The present invention provides a method of differentiating NK cells from CD14-positive mononuclear cells, comprising the step of mixing IL-15 and IL-21 with cord blood-derived CD14 positive mononuclear cells.

The umbilical cord blood-derived CD14 positive mononuclear cells are preferably prepared by the following steps, but are not limited thereto.

1) preparing CD3 ^- CD34 ^- CD56 ^- CD19 ^- cells by removing CD3, CD34, CD56 and CD19 - positive cells from cord blood - derived mononuclear cells; And

2) step 1) of CD3 ^- CD34 ^- CD56 ^- CD19 ^- CD14-negative cells by removing the cell from a CD3 ^- CD34 ^- CD56 ^- CD19 ^- one preferable that the step for producing the CD14 ⁺ cells, but not always limited thereto.

In one aspect of the present invention, in order to prepare CD3 ^- CD34 ^- CD56 ^- CD19 ^- CD14 ⁺ cells, a mononuclear cell layer (MNC layer) is separated from cord blood and then erythrocytes are removed to obtain mononuclear cells , And CD3 microbeads (Miltenyi Biotech) were added to induce magnetization of CD3-positive cells, which were then passed through a MACS column to isolate CD3-negative cells. Then, CD34, CD 56 and CD19 positive cells were removed from the CD3 negative mononuclear cells, and then CD14 microbeads, which are monocytic cell markers, were added and reacted to obtain CD14 positive mononuclear cells. As a result, as shown in Figs. 1 and 2, the recovery rate of CD14-positive cells was 4.4% and the purity was 84.6% (see Figs. 1 and 2).

In one aspect of the present invention, CD14-positive cells were analyzed by FACS using the respective CD15, CD19, CD33, CD56, CD117 and CD122 markers in order to confirm the expression of CD14-positive cells, CD56-negative cells, and all the CD14-positive cells showed phenotypes that were CD33-positive and some were partially positive with CD15, CD117, and CD122 (see FIG. 3).

In one aspect of the invention, CD34-positive hematopoietic stem cells and CD14-positive mononuclear cells were cultured in human IL-15, IL-21 and hydrocortisone medium, respectively, in order to compare the differentiation from CD14- As a result, NK differentiation degree of CD14-positive cells was significantly increased in a short period of time compared to that of CD34-positive cells (see FIGS. 4 and 5).

Therefore, it was confirmed that the method of differentiating NK cells from CD14-positive cells can obtain a large number of NK cells in a short period of time as compared with a method of differentiating NK cells from hematopoietic stem cells.

The present invention also provides a method for proliferating NK cells from CD14-positive mononuclear cells, comprising culturing cord blood-derived CD14 positive mononuclear cells with IL-15 and IL-21.

In one aspect of the present invention, CD3, CD34, CD56 and CD19 positive T cells, B cells and NK cells were removed from mononuclear cells isolated from cord blood to obtain CD14 positive cells. As a result, the recovery was 4.4% and the purity was 84.6 % (See Figs. 1 and 2).

In one aspect of the present invention, the expression of CD14-positive cells was analyzed by FACS. As a result, CD14-positive cells were CD19 and CD56-negative cells, and all of the CD14-positive cells exhibited CD33-positive phenotypes. CD122 and partially positive (see Fig. 3).

In one aspect of the present invention, CD34-positive hematopoietic stem cells and CD14-positive mononuclear cells include IL-15 and IL-21, which are known to promote NK differentiation and promote activity, in order to confirm the proliferation of NK cells from CD14- Fold induction of CDK-positive cells in the NK differentiation medium revealed that CD34-positive cells showed a poor proliferation rate in the NK differentiation medium, whereas CD14-positive cells increased the proliferation rate more than 2-fold (Fig. 6 Reference).

Therefore, it was confirmed that the method of proliferating NK cells from CD14-positive cells can obtain a large number of NK cells in a short period of time as compared with a method of proliferating NK cells from hematopoietic stem cells.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer comprising NK cells prepared by the method according to the present invention as an active ingredient.

The cancer is preferably any one selected from the group consisting of breast cancer, melanoma, gastric cancer, liver cancer, colon cancer and lung cancer, but is not limited thereto.

In one aspect of the present invention, CD3, CD34, CD56 and CD19 positive T cells, B cells and NK cells were removed from mononuclear cells isolated from cord blood to obtain CD14 positive cells. As a result, the recovery was 4.4% and the purity was 84.6 % (See Figs. 1 and 2).

In one aspect of the present invention, the expression of CD14-positive cells was analyzed by FACS. As a result, CD14-positive cells were CD19 and CD56-negative cells, and all of the CD14-positive cells exhibited CD33-positive phenotypes. CD122 and partially positive (see Fig. 3).

In one aspect of the present invention, NK cells (92% differentiation) differentiated from CD34-positive hematopoietic stem cells for 28 days and NK cells differentiated from CD14-positive mononuclear cells for 12 days The NK killing activity of CD34-derived NK cells differentiated for 28 days and that of CD14-derived NK cells differentiated for 14 days were similar to each other by the ⁵¹ Cr release assay with differentiation degree of 90% It was confirmed that NK cells derived from CD14-positive cells had a short-term high cytolytic activity as compared to CD34-skinned NK cells (see FIG. 7).

Therefore, it was confirmed that the differentiation of CD14-positive cells into NK cells which are active is more efficient than that of CD34 hematopoietic stem cells, since more numbers can be obtained.

The composition of the present invention may further contain at least one active ingredient which exhibits the same or similar function to the NK cell. For administration, one or more additional pharmaceutically acceptable carriers may be prepared. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, rings, granules or injection solutions. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically), and the dose may be determined depending on the body weight, age, sex, health condition, diet, The range varies depending on the excretion rate and severity of the disease. The daily dose of the composition according to the present invention is 0.01 to 5000 mg / kg, preferably 0.01 to 10 mg / kg, more preferably administered once to several times a day.

The present invention also provides a method of treating cancer comprising administering to a subject having a cancer a composition according to the present invention in a pharmaceutically effective amount.

In addition, the present invention provides a method for preventing cancer comprising administering to a subject a composition according to the present invention in a pharmaceutically effective amount.

The cancer is preferably any one selected from the group consisting of breast cancer, melanoma, gastric cancer, liver cancer, colon cancer and lung cancer, but is not limited thereto.

The composition of the present invention may further contain at least one active ingredient which exhibits the same or similar function to the NK cell. For administration, one or more additional pharmaceutically acceptable carriers may be prepared.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically), and the dose may be determined depending on the body weight, age, sex, health condition, diet, The range varies depending on the excretion rate and severity of the disease. The daily dose of the composition according to the present invention is 0.01 to 5000 mg / kg, preferably 0.01 to 10 mg / kg, more preferably administered once to several times a day.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

< Example  1> from cord blood Of hematopoietic stem cells  Isolation and Culture

The umbilical cord blood was diluted 2: 1 with RPMI 1640, and the umbilical cord blood was carefully placed on the upper part of the Ficoll-Paque. Then, the cord blood was centrifuged at 20,000 rpm for 30 minutes, After obtaining a mononuclear cell layer (MNC layer), erythrocytes were removed from the carefully taken cells to obtain mononuclear cells. Markers of hematopoietic stem cells After labeling with CD34 microbeads, CD34 ⁺ cells were isolated using MS / RS column and MACS.

As a result, as shown in FIG. 1 and FIG. 2, the recovery rate of CD34-positive cells was 0.6% and the purity was 81.1% (FIGS. 1 and 2).

< Example  2> Cord blood From mononuclear cells CD14  Isolation of positive cells

The umbilical cord blood was diluted 2: 1 with RPMI 1640, and the umbilical cord blood was carefully placed on the top of the Ficoll-Paque. Then, the cord blood was centrifuged at 20,000 rpm for 30 minutes, After obtaining a mononuclear cell layer (MNC layer), erythrocytes were removed from the carefully taken cells to obtain mononuclear cells. CD3 microbeads (Miltenyi Biotech) was added to the mononuclear cells as a T cell marker and reacted at 4 ° C for 30 minutes. Then, the cells were washed, suspended in a MACS buffer, and subjected to CD column analysis using Vario MACS. To obtain CD3 negative cells. CD34, CD56 and CD19-labeled microbeads were then added to the CD3-negative mononuclear cells to remove CD34, CD 56 and CD19-positive cells, followed by addition of CD14 microbeads as mononuclear cell markers And CD14 positive mononuclear cells were obtained using MS / RS column and MACS. The thus obtained CD14 positive cell purity was confirmed by flow cytometry (FACS).

As a result, as shown in Figs. 1 and 2, the recovery rate of CD14-positive cells was 4.4% and the purity was 84.6% (Fig. 1 and Fig. 2).

< Experimental Example  1> CD14  Expression analysis of benign cells

CD14-positive cells isolated from cord blood mononuclear cells (MNC) isolated in Example 2 were analyzed by FACS using respective CD15, CD19, CD33, CD56, CD117 and CD122 markers.

As a result, as shown in Fig. 3, CD14 positive cells were CD19 and CD56 negative cells, and all of the CD14 positive cells showed a phenotype that was CD33 positive, and some were partially positive with CD15, CD117 and CD122 3).

< Experimental Example  2> CD34  Positive cells and CD14  From benign cells NK  Comparison of cell differentiation

CD34-positive hematopoietic stem cells and CD14-positive mononuclear cells isolated from each of Example 1 and Example 2 were stained with CD3 and CD56 antibodies to determine NK differentiation (ratio of CD3 ^- CD56 ⁺ NK cell group) to FACS Respectively. Specifically, the CD34-positive hematopoietic stem cells and CD14-positive monocytes in each 12-well plate (Falcon) 1 × 10 ⁶ cells / ml human IL-15 with a concentration of (10 ng / ml, PeproTech) and IL-21 (10 ng / ml, PeproTech) and hydrocortisone ^{(hydrocortisone) (10 -6 M,} stem cell Tech.) is added to the Myelocult (Stem cell Technology) 37 ℃ using complete medium, at 5% CO ₂ 14 days or 12 days . When the cell concentration was over 2 x 10 ⁶ cells / well during the culturing, the cells were distributed using the medium of the initial used condition. 4, 8, 14, 18, and 21 days, respectively. On days 7 and 12, the cells were stained with CD3, CD122, and CD56 antibodies and analyzed for NK differentiation by FACS analysis.

As a result, as shown in FIG. 4 and FIG. 5, the NK differentiation degree (CD3 ^- CD56 ⁺ ) of CD34-positive cells at day 7 was 13.5% and the NK differentiation degree of CD14-positive cells was 78.5% NK differentiation (CD3 ^- CD56 ⁺ ) was found to be 41.3% and the NK differentiation degree of CD14 - positive cells was 90.2%. Therefore, it was confirmed that the NK differentiation degree of CD14-positive cells in the short term was significantly increased compared to CD34-positive cells (FIGS. 4 and 5).

< Experimental Example  3> CD34  Positive cells and CD14  From benign cells NK  Comparison of cell proliferation

 The CD34-positive hematopoietic stem cells and CD14-positive mononuclear cells prepared in Example 1 and Example 2 were cultured in NK differentiation medium containing IL-15 and IL-21, which are known to promote NK differentiation and promote activity, Cells were counted at 1, 4, 6, 7 and 12 days after culturing, respectively, and fold induction was examined.

As a result, as shown in FIG. 6, it was confirmed that the CD34-positive cells had a poor proliferation rate in the NK differentiation medium, whereas the CD14-positive cells increased the proliferation rate more than twice (FIG. 6).

< Experimental Example  4> CD34  Positive cells and CD14  From benign cells NK  Cell activity comparison

The ⁵¹ Cr release assay was performed on NK cells differentiated from CD34 positive hematopoietic stem cells for 28 days (92% differentiation degree) and NK cells differentiated from CD14 positive mononuclear cells (90% differentiation) for 14 days to determine cytolytic activity Respectively. Specifically, K562 cells, an NK-sensitive target, were labeled with radioactive isotope ⁵¹ Cr at 37 ° C for 1 hour and then washed. NK cells differentiated in vitro were washed and plated on a 96 well round bottom plate with ⁵¹ Cr-labeled K562 cells (10 ⁴ / well) as target cells according to the effector: target ratio. Lt; / RTI &gt; After 4 hours, 100 μl of culture supernatant was taken and the radioactivity was measured by γ-counter. The lysis (%) was calculated as (sample release-spontaneous / maximum release-spontaneous release) × 100.

As a result, as shown in FIG. 7, the NK killing activity of CD34-derived NK cells differentiated for 28 days and CD14-derived NK cells differentiated for 14 days at an E / T ratio of 10: , It was confirmed that NK cells derived from CD14-positive cells had a short-term high cytolytic activity compared to CD34-skinned NK cells. Therefore, it was confirmed that the differentiation from CD14-positive cells to NK cells which are active is more efficient than that of CD34 hematopoietic stem cells in a shorter period of time and a larger number can be obtained (Fig. 7).

Claims (5)

A method for differentiating NK cells from CD14-positive mononuclear cells, comprising the step of mixing CD14-positive mononuclear cells derived from cord blood with IL-15, IL-21 and hydrocortisone.
A method of propagating NK cells from CD14-positive mononuclear cells, comprising culturing cord blood-derived CD14-positive mononuclear cells with a mixture of IL-15, IL-21 and hydrocortisone.
3. The method according to claim 1 or 2, wherein the CD14 positive mononuclear cells are
1) preparing CD3 ^- CD34 ^- CD56 ^- CD19 ^- cells by removing CD3, CD34, CD56 and CD19 - positive cells from cord blood - derived mononuclear cells; And
Characterized in that the prepared by a process comprising the step of preparing a CD14 ^{+ cell-2)} step 1) CD3 of ^- Removal of the CD14 negative cells from the cell CD3 ^{- -} CD34 ^- CD56 ^- CD19 CD34 ^- CD56 ^- CD19 .


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