WO2009017274A1

WO2009017274A1 - An agent for differentiating hematopoietic stem cell into natural killer cell comprising yc-1 or il-21 and a method of differentiating hematopoietic stem cell into natural killer cell using thereof

Info

Publication number: WO2009017274A1
Application number: PCT/KR2007/004816
Authority: WO
Inventors: Inpyo Choi; Sukran Yoon; Sohyun Yun; Jin Woong Chung
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date: 2007-08-02
Filing date: 2007-10-02
Publication date: 2009-02-05
Anticipated expiration: 2010-02-02
Also published as: KR20090013602A; US8318491B2; KR100902340B1; US20110033415A1; CN101801374A; CN101801374B

Abstract

The present invention relates to an agent for differentiating hematopoietic stem cells into natural killer cells and a method for the differentiation, more precisely an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3- ( 5 ' - hydroxymethyl-2 ' -furyl) -1-benzylindazole] or IL-21 (Interleukin-21) as an active ingredient and a method for differentiating hematopoietic stem cells into natural killer cells using the same. The YC-I and IL-21 regulate the differentiation of hematopoietic stem cells into natural killer cells and increase the killing activity of NK cells. Therefore, the agent for NK cell differentiation of the present invention can be effectively used for cell therapy for the treatment of cancer by regulating the differentiation of hematopoietic stem cells into natural killer cells having tumor cell killing activity.

Description

[DESCRIPTION]

[invention Title]

AN AGENT FOR DIFFERENTIATING HEMATOPOIETIC STEM CELL INTO NATURAL KILLER CELL COMPRISING YC-I OR IL- 21 AND A METHOD OF DIFFERENTIATING HEMATOPOIETIC STEM CELL INTO NATURAL KILLER CELL USING THEREOF

[Technical Field]

The present invention relates to an agent for differentiating hematopoietic stem cells into natural killer cells and a method thereof, more precisely an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3- (5 ' -hydroxymethyl-2 ' - furyl) -1-benzylindazole] or IL-21 (Interleukin-21) as an active ingredient and a method for differentiating hematopoietic stem cells into natural killer cells using the same.

[Background Art] Hematopoietic stem cells, kind of adult stem cells, have a potential for differentiating into almost every blood forming cells (including erythrocytes, leucocytes, platelets and lymphocytes) and are constantly auto- regenerated as immune cells from the hematopoietic stem cell in bone marrow. The cells forming immune system, in particular natural killer cells (referred as "NK cells" hereinafter) , have an ability to kill tumor cells non- specifically .

This tumor cell killing activity of NK cells has been used for the treatment of a solid tumor using lymphokine activated killer cells (LAK) and tumor infiltration lymphocytes (TIL) or for the immune therapy via donor lymphocyte infusion (Itoh K, et al., J. Immunol. , 136: 3910-3915, 1986; Bordignon C, et al., Hematologia, 84: 1110-1149, 1999), which has drawn our attention as an advanced cell therapy for preventing rejection from bone marrow transplantation or organ transplantation. It was also reported that the defect in NK cell differentiation or activity is related to various diseases including 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) and lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002) . So, correction of NK cell functions seems to lead the way to treat these diseases. NK cells are derived from hematopoietic stem cells in bone marrow. The differentiation from hematopoietic stem cells into NK cells is composed of many steps, which have not been completely explained, yet. IL-21 (Interleukin-21) is a cytokine secreted by activated CD4+ T cells (Warren J., et al., Nature, 5: 688- 697, 2005) . IL-21 receptor (IL-21R) is expressed in lymphocytes such as dendrite cells, NK, T, and B cells (Takaki R., et al., J Immunol., 175: 2167-2173, 2005). The structure of IL-21 is very similar to those of IL-2 and IL- 15, and IL-21R shares γ-chain with IL-2R, IL-15, IL-7R or IL-4R (Ensminger SM, et al. , J Immunol. 167 (1) : 532-541, 2001) . According to the previous reports, IL-21 induces maturation of NK cell precursor in bone marrow (Parrish- Novak J., et al., Nature, 408: 57-63, 2000) and characteristically increases effector functions of NK such as cytokine generation ability and killing activity (Strengell M, et al. , J Immunol., 170: 5464-5469, 2003; Brady J, et al., J Immunol., 172: 2048-2058, 2004). IL-21 also increases effector functions of CD8+ T cells, leading to the promotion of anticancer response of innate or adaptive immune system (Takaki R., et al., J Immunol. , 175: 2167-2173, 2005; Moroz A., et al., J Immunol, 173: 900-909, 2004) . In addition, IL-21 activates NK cells separated from human peripheral blood (Parrish-Novak J., et al., Nature, 408: 57-63, 2000) and plays an important role in differentiation of NK cells from hematopoietic stem cells separated from umbilical cord blood (Sonia A. P, et al., Int. Immunol., 18: 49-58, 2006).

YC-I [3- (5 ' -hydroxymethyl-2 ' -furyl) -1-benzylindazole] was first developed as an agent for treating circulatory diseases after it was proved to inhibit coagulation of platelets and activate soluble GC (guanylyl cyclase) to inhibit contraction of blood vessel (Ko FN, et al., Blood 84. No. 12:4226-4233 1994; Teng CM, et al., Eur J Pharmacol, 320:161-6, 1997; Galle J., et al., Br J Pharmacol, 127: 195-203, 1999) . It was additionally found in recent studies that it inhibits HIF-lα accumulation induced in hypoxia, reduces expressions of HIF-I target genes (VEGF, erythropoietin, etc.) (Chun YS, et al., Biochem Pharmacol, 61:947-954, 2001), and inhibits tumor growth and angiogenesis in animals (Yeo EJ, et al., J Natl Cancer Inst, 95: 516-525, 2003; Pan SL, et al., J pharmacol Exp Ther, 314:35-42, 2005). So, YC-I becomes a leading compound for the development of an anticancer agent targeting HIF-lα (Yeo EJ, et al., Cancer Res, 66: 6345-6352, 2006). Even though YC-I is very useful for the study of HIF-lα (Moeller BJ, et al., Cancer cell 5: 429-441, 2004; Funasaka T, et al., FASEB J, 19:1422-1430, 2005), the mechanism of anticancer activity of YC-I has not been disclosed, yet (Sun. H. L., et. al. Oncogene 1-11, 2006). The present inventors completed this invention by confirming that IL-21 (Interleukin-21) and YC-I [3-(5'- hydroxymethyl-2 ' -furyl) -1-benzylindazole] promote the differentiation of hematopoietic stem cells into natural killer cells and increase the killing activity of natural killer cells.

[Disclosure] [Technical Problem]

It is an object of the present invention to provide an agent for differentiating hematopoietic stem cells into natural killer cells and a method for the differentiation.

[Technical Solution]

To achieve the above object, the present invention provides an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3- (5'- hydroxymethyl-2 ' -furyl) -1-benzylindazole] as an active ingredient.

The present invention also provides an anticancer agent comprising IL-21 (Interleukin-21) as an active ingredient .

The present invention further provides an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3- (5 ' -hydroxymethyl-2 '- furyl) -1-benzylindazole] and IL-21 (Interleukin-21) as active ingredients.

The present invention also provides an anticancer agent comprising YC-I [3- (5 ' -hydroxymethyl-2 ' -furyl) -1- benzylindazole] and IL-21 (Interleukin-21) as active ingredients .

The present invention also provides a method for differentiating hematopoietic stem cells into natural killer cells using IL-21 and/or YC-I.

The present invention also provides a cell therapy method for the treatment of cancer, containing the step of administering natural killer cells differentiated by the above method to tumor cells. The present invention also provides a method for increasing the killing activity of NK cells, containing the step of administering IL-21 (Interleukin-21) to hematopoietic stem cells.

The present invention also provides a method for increasing the killing activity of NK cells, containing the step of administering YC-I [3- (5 ' -hydroxymethyl-2 ' -furyl) - 1-benzylindazole] to hematopoietic stem cells.

The present invention also provides a method for increasing the killing activity of NK cells, containing the step of administering YC-I [3- (5 ' -hydroxymethyl-2 ' -furyl) - 1-benzylindazole] and IL-21 (Interleukin-21) to hematopoietic stem cells.

The present invention also provides a cell therapy method for the treatment of cancer, containing the step of administering natural killer cells with the improved tumor cell killing activity to tumor cells.

The present invention also provides a use of YC-I for the preparation of an agent for differentiating hematopoietic stem cells into natural killer cells or an anticancer agent.

The present invention also provides a use of IL-21 for the preparation of an anticancer agent.

In addition, the present invention provides a use of YC-I and IL-21 for the preparation of an agent for differentiating hematopoietic stem cells into natural killer cells or an anticancer agent.

[Advantageous Effect]

YC-I and IL-21 are involved in NK cell differentiation and increase the killing activity of NK cells. So, an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I or IL-21 as an active ingredient and an anticancer agent comprising IL- 21 as an active ingredient can be effectively used for the development of a novel method for cell therapy for cancer and for the regulation of NK cell differentiation.

[Description of Drawings] The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Fig. 1 is a graph illustrating the purity of CD34⁺ hematopoietic stem cells measured by flow cytometry (the number written in quadrant indicates the purity) .

Fig. 2 is a schematic diagram illustrating the process of the differentiation of hematopoietic stem cells separated from human cord blood into mature NK (mNK) cells through NK precursors (pNK) :

HSC: hematopoietic stem cells; SCF: stem cell factor; and Flt3L: FMS-like tyrosine kinase 3 ligand. Fig. 3 is a graph illustrating the expression of the surface molecule of each NK cell differentiation stage (the number written in quadrant indicates the purity) .

Fig. 4 is a graph illustrating the result of FACS. In differentiation of NK cells, IL-21 was treated to pNKs (NK precursor cells), which were cultured in the presence of IL-15 until they became differentiated into mature NK cells, followed by examination by FACS.

Fig. 5 is a graph illustrating the population of

CD56+ NK cells over differentiation time, which is represented by %. Precisely, in NK differentiation, IL-21 was treated to pNKs, which were cultured in the presence of

IL-15 and CD56+ NK cells were counted over cultivation time.

Fig. 6 is a graph illustrating the result of ⁵¹Cr release assay. In NK differentiation, IL-21 was treated to pNKs, which were cultured in the presence of IL-15 until they became differentiated into mature NK (mNK) cells, followed by ⁵¹Cr release assay:

E:T: effector cell : target cell.

Fig. 7 is a graph illustrating the result of FACS analysis. In NK differentiation, YC-I was treated to pNKs, which were cultured in the presence of IL-15 until they became differentiated into mature NK (mNK) cells, followed by FACS.

Fig. 8 is a graph illustrating the population of CD56+ NK cells. Precisely, in NK differentiation, YC-I was treated to pNKs, which were cultured in the^' presence of IL- 15. CD56+ NK cells were counted over the cultivation time and represented by %.

Fig. 9 is a graph illustrating the result of ⁵¹Cr release assay. In NK differentiation, YC-I was treated to pNKs, which were cultured in the presence of IL-15 until they became differentiated into mature NK (mNK) cells, followed by ⁵¹Cr release assay:

E: T: effector cell: target cell. Fig. 10 is a graph illustrating the result of FACS analysis. In NK differentiation, both IL-21 and YC-I were treated to pNKs, which were cultured in the presence of IL- 15 until they became differentiated into mature NK (mNK) cells, followed by FACS. Fig. 11 is a graph illustrating the population of CD56+ NK cells over cultivation time. In NK differentiation, both IL-21 and YC-I were treated to pNKs, which were cultured in the presence of IL-15, followed by measuring the population of CD56+ NK cells over cultivation time.

[Mode for Invention]

Hereinafter, the present invention is described in detail. The present invention provides an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3- (5 ' -hydroxymethyl-2 ' - furyl) -1-benzylindazole] as an active ingredient.

The YC-I(Ci₉Hi₆N₂O₂) is represented by formula 1. [Formula l]

The IL-21 (Interleukin-21) is preferably selected from the group consisting of (a) the protein comprising the amino acid sequence represented by SEQ. ID. NO: 1; (b) the protein encoded by the DNA containing the coding region of the nucleotide sequence represented by SEQ. ID. NO: 2; (c) the protein comprising the amino acid sequence with substitution, deletion, insertion and/or addition of one or more amino acids in the amino acid sequence represented by SEQ. ID. NO: 1 and is functionally equal to the protein comprising the amino acid sequence represented by SEQ. ID. NO: 1; and (d) the protein encoded by the DNA hybridized with the DNA comprising the nucleotide sequence represented by SEQ. ID. NO: 2 under the strict condition and is functionally equal to the protein comprising the amino acid sequence represented by SEQ. ID. NO: 1.

Hybridization under the strict condition enables the selection of DNA having the nucleotide sequence with high homology. Thus, the chances are high for the separated protein therefrom to be a protein that is functionally equal to IL-21. The nucleotide sequence with high homology means, for example, the nucleotide sequence having at least 70% homology with the nucleotide sequence represented by SEQ. ID. NO: 2, preferably having at least 80% homology and more preferably at least 90% and most preferably at least 95% homology with the nucleotide sequence represented by SEQ. ID. NO: 2. In the case of amino acid sequence, the amino acid sequence having at least 70% homology, preferably at least 80% homology, more preferably at least 90% and most preferably at least 95% homology with the amino acid sequence represented by SEQ. ID. NO: 1 can be selected. The percent of homology can be determined by the conventional algorithm selected by those in the art. The hybridization can be performed by DNA-DNA hybridization under the strict condition (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridization, IRL Press, U.K.) known to those in the art and the condition can be determined in the washing process after hybridization. The present invention further provides an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3- (5 ' -hydroxymethyl-2 ' - furyl) -1-benzylindazole] and IL-21 (Interleukin-21) as active ingredients.

The present inventors separated CD34+ hematopoietic stem cells (referred as "HSC cells" hereinafter) with 92% purity from cord blood (see Fig. 1) . The HSC cells were differentiated into mature NK cells through pNKs (see Fig. 2) and the expression of the surface molecule was examined for each NK cell differentiation stage (see Fig. 3) . As a result, CD56 cells were increased.

In NK cell differentiation, IL-21 was treated to pNKs, which were cultured in the presence of IL-15 to differentiate pNKs into mNK cells. FACS analysis was performed. As a result, CD56+ NK cell population was increased in IL-21 treated group, compared with the IL-21 non-treated control (treated with IL-15 alone) (see Figs. 4 and 5) , suggesting that IL-21 was involved in NK differentiation. From the result of ⁵¹Cr release assay, it was confirmed that IL-21 treated group exhibited increased killing activity, compared with IL-21 non-treated group

(treated with IL-15 alone) (see Fig. 6) , suggesting that

IL-21 was involved in the activation of the killing activity of NK cells. NK precursor cells (pNK) were treated with YC-I and cultured in the presence of IL-15 to differentiate into matured NK (mNK) cells, followed by FACS analysis. As a result, CD56+ NK cells were increased in YC-I treated group, compared with YC-I non-treated group (treated with IL-15 alone) (see Figs. 7 and 8), suggesting that YC-I was involved in NK cell differentiation. From the result of ⁵¹Cr release assay, it was confirmed that YC-I treated group exhibited increased killing activity, compared with IL-21 non-treated group (treated with IL-15 alone) (see Fig. 9) , suggesting that YC-I was involved in the activation of the killing activity of NK cells.

NK precursor cells (pNK) were treated with both IL-21 and YC-I at the same time and NK cell differentiation was investigated. As a result, NK cell differentiation was significantly increased (see Figs. 10 and 11).

As explained hereinbefore, IL-21 and YC-I promote differentiation from hematopoietic stem cells into natural killer cells and increase the killing activity of NK cells, so that they can be effectively used as agents for differentiating hematopoietic stem cells into NK cells.

The agent for differentiating into NK cells and the anticancer agent of the invention can be used for cell therapy for the treatment of cancer.

The cancer herein is preferably selected from the group consisting of breast cancer, melanoma, stomach cancer, liver cancer, blood cancer, colon cancer, and lung cancer, but not always limited thereto. If there is a defect in the differentiation and activity of NK cells, various cancers might be developed, for example, breast cancer (Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), melanoma (Ryuke Y, et al., Melanoma Res., 2003, 13: 349-356) and lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002), etc, according to the previous reports. Therefore, it is expected to treat such cancers as the above by regulating NK cell differentiation using the agent for NK cell differentiation and the anticancer agent of the invention. The agent for NK cell differentiation and the anticancer agent of the present invention can be administered orally or parenterally and be used in general forms of pharmaceutical formulation. The agent for NK cell differentiation and the anticancer agent of the present invention can be prepared for oral or parenteral administration by mixing with generally used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients. Solid formulations for oral administration are tablets, pills, dusting powders, granules and capsules. These solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water- insoluble excipients, suspensions, emulsions, and suppositories. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc. Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin, etc. The effective dosage of the agent for NK cell differentiation or the anticancer agent of the present invention is 0.1 ~ 0.2 mg/kg, and more preferably 0.15 mg/kg, and the administration times are preferably 1~3 times a day.

Particularly, the method for differentiating hematopoietic stem cells into NK cells comprises the following steps:

1) inducing proliferation of NK precursor cells by adding a NK precursor inducer to hematopoietic stem cells; and 2) differentiating the NK precursor cells into mature NK cells by adding IL-21 and/or YC-I to the NK precursor cells of step 1) .

In step 1) , the "NK precursor inducer" means any substance that is able to induce differentiation from hematopoietic stem cells into NK precursor cells, which is preferably SCF or Flt3L, but not always limited thereto.

In step 2) , the preferable treatment amount of IL-21 is 10 ng/m-β ~ 50 nq/ml, and the treatment amount of YC-I is 0.5 uM ~ 5 uM. In the case of co-treatment of YC-I and IL- 21, preferable contents of YC-I and IL-21 are respectively 0.5 uM and 10 nq/ml, but not always limited thereto.

In step 2), the NK precursor cells are preferably cultured with IL-15 (Interleukin-15) , but not always limited thereto.

According to the conventional method, hematopoietic stem cells separated from cord blood are treated with IL-15 and IL-21 simultaneously to induce proliferation and differentiation at the same time. So, differentiation speed is very slow and thus takes long time (30 days) . However, in the method of the present invention, differentiation from hematopoietic stem cells into NK precursor cells is first induced by adding SCF and Flt3L and then differentiation into mature NK cells is induced by adding IL-15 and IL-21 and/or YC-I, which makes the method efficient and reduces differentiation time (14 days) .

The present invention also provides a cell therapy method for the treatment of cancer, containing the step of administering natural killer cells differentiated by the above method to tumor cells.

The differentiation from hematopoietic stem cells into NK cells above is induced in vitro.

The cancer herein is preferably selected from the group consisting of breast cancer, melanoma, stomach cancer, liver cancer, blood cancer, colon cancer, and lung cancer, but not always limited thereto.

To induce differentiation from hematopoietic stem cells into NK cells, YC-I or IL-21 can be administered but co-administration of YC-I and IL-21 is preferred.

The present invention also provides a method for increasing the killing activity of NK cells, containing the step of administering IL-21 (Interleukin-21) to hematopoietic stem cells.

The effective dosage of IL-21 is 10 ng/ml ~ 50 ng/m£, and is preferably administered together with IL-15 (Interleukin-15) for the culture, but not always limited thereto .

The present invention also provides a method for increasing the killing activity of NK cells, containing the step of administering YC-I [3- (5 ' -hydroxymethyl-2 ' -furyl) - 1-benzylindazole] to hematopoietic stem cells. The preferable dosage of YC-I is 0.5 uM ~ 5 uM, and is preferably administered together with IL-15 (Interleukin-15) for the culture, but not always limited thereto . The present invention also provides a method for increasing the killing activity of NK cells, containing the step of administering YC-I [3- (5 ' -hydroxymethyl-2 ' -furyl) -

1-benzylindazole] and IL-21 (Interleukin-21) to hematopoietic stem cells.

The preferable hematopoietic stem cells for the invention are those who are under-going differentiation into NK cells.

The preferable doses of YC-I and IL-21 are respectively 0.5 uM and 10 ng/ml., which are preferably co- treated with IL-15 (Interleukin-15) for the culture of hematopoietic stem cells, but not always limited thereto.

The killing activity of NK cells is increased in vitro. The cancer herein is preferably selected from the group consisting of breast cancer, melanoma, stomach cancer, liver cancer, blood cancer, colon cancer, and lung cancer, but not always limited thereto. To increase the killing activity of NK cells, YC-I or IL-21 can be treated but co-administration of YC-I and IL- 21 is more preferred.

In addition, the present invention provides a use of YC-I or IL-21 for the preparation of an agent for differentiating hematopoietic stem cells into natural killer cells or an anticancer agent.

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Separation of hematopoietic stem cells from cord blood Cord blood provided from Department of Obstetric & Gynecology, Konyang University Hospital, Taejon, Korea, for the purpose of study only was diluted in RPMI 1640 (GIBCO- BRL, USA) at the ratio of 2:1. The prepared cord blood was loaded on the upper part of Ficoll-Paque (Sigma, USA), followed by centrifugation (20,000 rpm, 30 minutes) to separate mononuclear cells (MNC) . Erythrocytes were eliminated from the obtained cells and the obtained mononuclear cells were marked with the hematopoietic stem cell marker ^λCD34 microbeads' . Then, CD34+ cells were separated by MS/RS column and MACS (Magnetic Activated Cell Sorter) . The purity of the obtained CD34+ hematopoietic stem cells (referred as "HSC cells" hereinafter) was measured by FACS (BD Bioscience, Mountainview, CA) , which was 92% (Fig. 1) .

Example 2; Differentiation from hematopoietic stem cells of cord blood into NK cells

The HSC cells separated from cord blood in Example 1 were inoculated in a 12-well plate (Falcon, USA) containing the Myelocult complete medium (Stem cell Technology, CA) supplemented with 30 ng/m£ of human Factor (PeproTech, USA) , 50 ng/m# of human Flt3L (FMS-like tyrosine kinase 3 ligand, PeproTech, USA), 5 ng/mϋ. of human IL-7 (PeproTech, USA), and 10^~6 M of hydro SCF (Stem Cell hydrocortisone, Stem cell Technology, CA) at the concentration of IxIO⁶ cells/well, followed by culture for 14 days in a 37 ^°C, 5% CO₂ incubator. Three days later, half of the supernatant was replaced with a fresh medium containing cytokines having the same composition as the above. For the differentiation into mature NK cells (referred as "NK cells" hereinafter) , HSC cells were recovered 14 days later and cultured again for 14 days in the presence of human IL-15 (30 nq/mt, PeproTech, USA) . Three days later, half of the medium was replaced with a fresh medium containing cytokines having the same composition as the above (Fig. 2) . On the 28^th day of culture, the purity of NK cells was measured by using anti- CD56 antibody and the expression of NK cell receptor was measured by flow cytometry (FACS) (Fig. 3) .

Example 3: Effect on NK cell differentiation of IL-21

To investigate the effect on NK cell differentiation of IL-21 known to promote differentiation and increase activity, IL-21 (20 nq/ml, PeproTech, USA) was treated during the process of differentiation from hematopoietic stem cells of cord blood into mature NK cells (mNK) through NK precursor cells (pNK) . The cells were cultured in the presence of IL-15, followed by FACS analysis and ⁵¹Cr release assay.

IL-21 has the amino acid sequence represented by SEQ. ID. NO: 1 and the nucleotide sequence represented by SEQ. ID. NO: 2.

From the result of FACS analysis, it was confirmed that CD56+ NK cell population was increased in IL-21 treated group (Figs. 4 and 5) . From the result of ⁵¹Cr release assay, it was confirmed that the killing activity of NK cells was increased by IL-21 (Fig. 6). E:T (effector cell: target cell) was 2.5:1. The above results indicate that IL-21 is involved in NK cell differentiation and directly affects the killing activity of NK cells.

Example 4: Effect on NK cell differentiation of YC-I To investigate the effect on NK cell differentiation of YC-I [3- (5 ' -hydroxymethyl-2 ' -furyl) -1-benzylindazole, formula 1] known as an anticancer agent functioning in suppressing HIF-lα activity to inhibit tumor cell growth, YC-I (1 uM, Sigma) was treated during the process of differentiation from hematopoietic stem cells of cord blood into mature NK cells (mNK) through NK precursor cells (pNK) . The cells were cultured in the presence of IL-15, followed by FACS analysis and ⁵¹Cr release assay.

From the result of FACS analysis, it was confirmed that CD56+ NK cell population was increased in YC-I treated group (Figs. 7 and 8) .

From the result of ⁵¹Cr release assay, it was confirmed that the killing activity of NK cells was increased by YC-I (Fig. 9) . E:T (effector cell: target cell) was 5:1. The above results indicate that YC-I is involved in NK cell differentiation and directly affects the killing activity of NK cells.

Example 5: Synergy effect of IL-21 and YC-I on NK cell differentiation To investigate synergy effect of IL-21 and YC-I on

NK cell differentiation, IL-21 (10 ng/ml) and YC-I (0.5 uM) were co-treated during the process of differentiation from hematopoietic stem cells of cord blood into mature NK cells

(mNK) through NK precursor cells (pNK) . The cells were cultured in the presence of IL-15, followed by FACS analysis. At that time, the concentrations of IL-21 and YC-I were regulated low in order not to affect NK cell differentiation (10 nq/ml of IL-21 and 0.5 uM of YC-I). From the result of FACS analysis, NK cells were slightly increased when each of the two was separately treated but NK cells were significantly increased when both of them were treated together (Figs. 10 and 11), indicating that the co-treatment of IL-21 and YC-I brings synergy effect .

[industrial Applicability]

As explained hereinbefore, YC-I and IL-21 regulate differentiation of hematopoietic stem cells into natural killer cells and increase the killing activity of NK cells. Therefore, an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3- (5'- hydroxymethyl-2 ' -furyl) -1-benzylindazole] or IL-21 (Interleukin-21) as an active ingredient and a method for differentiating hematopoietic stem cells into natural killer cells using the same can be effectively used for cell therapy for the treatment of cancer.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims .

Claims

[CLAIMS]

[Claim l]

An agent for NK cell differentiation comprising YC-I [3- (5 ' -hydroxymethyl-2 ' -furyl) -1-benzylindazole] as an active ingredient.

[Claim 2]

The agent for NK cell differentiation according to claim 1, wherein the YC-I promotes the differentiation from hematopoietic stem cells into natural killer cells or increases the killing activity of NK cells.

[Claim 3]

An anticancer agent comprising IL-21 (Interleukin 21) as an active ingredient.

[Claim 4]

The anticancer agent according to claim 3, wherein the cancer is selected from the group consisting of breast cancer, melanoma, stomach cancer, liver cancer, blood cancer, colon cancer and lung cancer.

[Claim 5] An agent for NK cell differentiation comprising YC-I [3- (5^I-hydroxymethyl-2'-furyl) -1-benzylindazole] and IL-21 (Interleukin-21) as active ingredients.

[Claim β]

An anticancer agent comprising YC-I [3-(5'- hydroxymethyl-2 ' -furyl) -1-benzylindazole] and IL-21 (Interleukin-21) as active ingredients.

[Claim 7]

A method for differentiating hematopoietic stem cells into NK cells comprising the following steps:

1) inducing proliferation of NK precursor cells by adding a NK precursor inducer to hematopoietic stem cells; and

2) differentiating the NK precursor cells into mature NK cells by adding IL-21 and/or YC-I to the NK precursor cells of step 1) .

[Claim 8]

The method according to claim 7, wherein the NK precursor inducer of step 1) is SCF and Flt3L.

[Claim 9] The method according to claim 7, wherein the IL-21 of step 2) is a protein selected from the group consisting of: (a) the protein comprising the amino acid sequence represented by SEQ. ID. NO: 1; (b) the protein encoded by the DNA containing the coding region of the nucleotide sequence represented by SEQ. ID. NO: 2;

(c) the protein comprising the amino acid sequence with substitution, deletion, insertion and/or addition of one or more amino acids in the amino acid sequence represented by SEQ. ID. NO: 1 and is functionally equal to the protein comprising the amino acid sequence represented by SEQ. ID. NO: 1; and

(d) the protein encoded by the DNA hybridized with the DNA comprising the nucleotide sequence represented by

SEQ. ID. NO: 2 under the strict condition and is functionally equal to the protein comprising the amino acid sequence represented by SEQ. ID. NO: 1.

[Claim 10]

The method according to claim 7, wherein the dosage of the IL-21 of step 2) is 10 nq/mi ~ 50 nq/mt.

[Claim 11] The method according to claim 7, wherein the dosage of the YC-I of step 2) is 0.5 uM - 5 uM.

[Claim 12] The method according to claim I₁ wherein the NK precursor cells of step 2) are co-cultured with IL-15 (Interleukin-15) .

[Claim 13] A cell therapy method for the treatment of cancer containing the step of administering NK cells differentiated by the method of claim 7 to tumor cells.

[Claim 14] A method for increasing the killing activity of NK cells containing the step of administering IL-21 (Interleukin-21) to hematopoietic stem cells.

[Claim 15] A method for increasing the killing activity of NK cells containing the step of administering YC-I [3- (5'- hydroxymethyl-2 ' -furyl) -1-benzylindazole] to hematopoietic stem cells.

[Claim 16] A method for increasing the killing activity of NK cells, containing the step of administering YC-I [3- (5'- hydroxymethyl-2 ' -furyl) -1-benzylindazole] and IL-21 (Interleukin-21) to hematopoietic stem cells.

[Claim 17]

The method according to one of claim 14 - claim 16, wherein the hematopoietic stem cells are undergoing differentiation into NK cells.

[Claim 18]

The method according to one of claim 14 - claim 16, wherein the hematopoietic stem cells are co-cultured with IL-15 (Interleukin-15) .

[Claim 19]

A cell therapy method for the treatment of cancer containing the step of administering NK cells with improved killing activity by one of the method of claim 14 - claim 16 to tumor cells.

[Claim 20]

A use of YC-I for the preparation of an agent for NK cell differentiation or an anticancer agent. [Claim 2l]

A use of IL-21 for the preparation of an anticancer agent .

[Claim 22]

A use of YC-I and IL-21 for the preparation of an agent for NK cell differentiation or an anticancer agent.