KR101557343B1 - A pharmaceutical composition using a fusion protein comprising dna binding domain from gata-3 of transcription factors and protein transduction domain for treating an allergic asthma and a method using the same - Google Patents

Abstract

The present invention relates to a transcription factor inhibitor having a fusion protein comprising a DNA binding domain and a protein transduction domain of the GATA transcription factor family (GATA binding transcription factor family). The inhibitor according to the present invention can effectively treat a related disease caused by excessive activity by inhibiting a major transcription process using a recombinant fusion protein comprising a DNA binding domain and a protein transport domain (PTD) of a GATA- 3 transcription factor.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for treating allergic asthma using a protein fused with a DNA binding domain of a transcription factor GATA-3 and a protein transduction domain, and a therapeutic method using the protein. AND PROTEIN TRANSDUCTION DOMAIN FOR TREATING AN ALLERGIC ASTHMA AND METHOD USING THE SAME}

The present invention relates to a pharmaceutical composition for treating allergic asthma using a protein fused with a DNA binding domain of a transcription factor GATA-3 and a protein transport domain, and a therapeutic method using the same.

GATA-3 is a zinc finger structure protein composed of 370 amino acids and is one of GATA family proteins. Previous studies using the GATA-3 deficient animal model revealed that GATA-3 is a major transcription factor that regulates T helper 2 (TH2) cell differentiation both in vitro and in vivo. In addition, studies using GATA-3 transgenic animal models have shown that GATA-3 is selectively expressed in TH2 cells and the expression of TH2 cell-specific cytokines is greatly increased by overexpression of GATA-3. In addition, GATA-3 functions to regulate signal transduction pathways inside the cell, thereby maintaining the differentiation process of activated T cells into TH2 cells. In addition, recent studies have shown that regulatory T cells also express in regulatory T cells (Treg), which is an important transcription factor that inhibits the induction of disease by migrating to disease sites induced by TH2. GATA-3 has a DNA binding domain consisting of two zinc fingers at the carboxy terminus and a transactivation domain at the amino terminus. The DNA binding domain binds to the promoter of the desired gene of GATA-3 to promote transcription, and the transactivation domain further promotes transcription through binding with a cofactor.

GATA-3 enhances the immune response by TH2 cells through a self-activating mechanism that promotes the secretion of TH2 cell-specific cytokines IL-4, IL-5 and IL-13 while promoting self-expression. These immune responses play a very important role in eliminating pathogens from the outside. However, when the immune response by TH2 cells is not regulated more than necessary, it causes autoimmune disease do. Representative autoimmune diseases caused by excessive immune response of TH2 cells include allergic asthma, atopic dermatitis, systemic lupus erythematosus, ulcerative colitis, pelargatitis. Until now, treatment of these diseases by reducing the overall inflammatory response has not been able to specifically eliminate the etiology depending on the type of disease, which may result in lower treatment efficiency or other side effects.

Protein transduction domains (PTDs) are known to effectively transfer physiologically active molecules such as proteins, DNAs, and RNAs that are fused together into short peptides with high hydrophobicity. The inventors of the present invention have developed two PTDs and registered the patent. Since the protein transport domain can transport physiologically active molecules as well as the cytoplasm, it has properties suitable for transferring the modified transcription factor, which is the core substance of the present invention, into the nucleus.

A recombinant fusion protein having only the DNA binding domain of GATA-3, which is the main transcription factor of TH2 cells, is prepared and transferred into cells, whereby the recombinant fusion protein binds to the desired promoter in place of wild-type GATA-3 in the cell . Since this recombinant fusion protein has only a DNA binding site without a transactivation domain, it is a dominant negative mutant that can bind to the desired promoter but can not promote transcription. Therefore, the recombinant fusion protein can not bind to the wild type GATA-3 It acts as a competitive inhibitor. In this way, the inventors of the present invention have completed the present invention by obtaining a result that effectively inhibits the autoimmune disease caused by excessive activity of TH2 cells by inhibiting the activity of TH2 cells.

A transcription factor inhibitor in which a DNA binding domain and a protein transduction domain of a GATA-3 transcription factor are fused and a method for producing the same.

It is also an object of the present invention to provide a recombinant fusion protein comprising a DNA binding domain and a protein transport domain of a GATA-3 transcription factor, a nucleic acid encoding the same, a vector comprising the nucleic acid, and a host cell containing the vector.

Also provided is a pharmaceutical composition for treating a transcription factor-related disease comprising a recombinant fusion protein comprising a DNA binding domain and a protein transduction domain of a GATA-3 transcription factor and a pharmaceutically acceptable excipient and a DNA binding domain and a protein transport domain And a pharmaceutically acceptable excipient. ≪ Desc / Clms Page number 2 >

It is also an object of the present invention to provide a method for treating a transcription factor related disease comprising administering to a patient having a transcription factor related disease an effective amount of a recombinant fusion protein comprising a DNA binding domain and a protein transduction domain of a GATA-3 transcription factor have.

To accomplish this object, in one embodiment, there is provided a transcription factor inhibitor having a fusion protein comprising a DNA binding domain and a protein transduction domain of a GATA transcription factor family. In another embodiment, the transcription factor is any one selected from the group consisting of GATA-1, GATA-2, GATA-3, GATA-4, GATA-5 and GATA-6. In another embodiment, there is provided a transcription factor inhibitor characterized in that the transcription factor is GATA-3. In another embodiment, the protein transport domain is selected from the group consisting of Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia), Pep- CTP (Cytoplamic transduction peptide). The present invention also provides a transcription factor inhibitor. In another embodiment, there is provided a transcription factor inhibitor characterized in that the protein transport domain has the sequence represented by SEQ ID NO: 1. In another embodiment, there is provided a transcription factor inhibitor characterized in that the DNA binding domain of the transcription factor has the sequence represented by SEQ ID NO: 2. In another embodiment, the fusion protein has a sequence represented by SEQ ID NO: 3.

In one embodiment, a method is provided for preparing a fusion protein comprising a DNA binding domain and a protein transport domain of a GATA binding transcription factor family. In another embodiment, the transcription factor is any one selected from the group consisting of GATA-1, GATA-2, GATA-3, GATA-4, GATA-5 and GATA-6. do. In another embodiment, there is provided a method of preparing a fusion protein characterized in that the transcription factor is GATA-3t. In another embodiment, the protein transport domain is selected from the group consisting of Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia), Pep- And CTP (Cytoplamic transduction peptide). The present invention also provides a method for producing a fusion protein. In another embodiment, the present invention provides a method for producing a fusion protein, wherein the protein transport domain has the sequence represented by SEQ ID NO: 1. In another embodiment, the DNA binding domain of the transcription factor has the sequence of SEQ ID NO: 2. In another embodiment, the fusion protein has a sequence represented by SEQ ID NO: 3.

In one embodiment, there is provided a pharmaceutical composition for treating an autoimmune disease comprising a fusion protein comprising a DNA binding domain and a protein transport domain of a GATA binding transcription factor family and a pharmaceutically acceptable excipient . In another embodiment, the pharmaceutical composition is characterized in that the transcription factor is any one selected from the group consisting of GATA-1, GATA-2, GATA-3, GATA-4, GATA-5 and GATA-6. In another embodiment, there is provided a pharmaceutical composition, wherein the transcription factor-related disease is a skin disease when the transcription factor is GATA-3 (GATA binding protein 3). In another embodiment, the autoimmune disease is any one of allergic asthma, atopic dermatitis, systemic lupus erythematosus, ulcerative colitis, and pelargatitis. In another embodiment, the protein transport domain is selected from the group consisting of Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia), Pep- CTP (cytoplasmic transduction peptide). The present invention also provides a pharmaceutical composition for treating a transcription factor-related disease. In another embodiment, the present invention provides a pharmaceutical composition for treating a transcription factor-related disease, wherein the protein transport domain has the sequence represented by SEQ ID NO: 1. In another embodiment, the present invention provides a pharmaceutical composition for treating a transcription factor-related disease, wherein the DNA binding domain of the transcription factor has the sequence represented by SEQ ID NO: 2. In another embodiment, there is provided a pharmaceutical composition for treating a transcription factor-related disease, wherein the fusion protein has the sequence represented by SEQ ID NO: 3.

In the present invention, the "transcription factor" includes, but is not limited to, a DNA binding domain-containing factor that binds to the promoter region of DNA and regulates transcription. For example, the GATA binding transcription factor family GATA-1, GATA-2, GATA-3, GATA-4, GATA-5 and GATA-6.

In the present invention, the term "protein transduction domain" includes, but is not limited to, a protein consisting of 7 to 50 amino acids and capable of delivering DNA or RNA into cells as well as a protein having a size of 120 kDa or more, , Hph-1, Mph-1 and Sim-2.

In the present invention, the term "autoimmune disease" refers to a disease in which an immune cell to be protected from an intruder such as a bacterium, a virus or a foreign object attacks his or her body, including but not limited to allergic asthma, atopic dermatitis, Leucosis, ulcerative colitis, and pelargatitis.

The present invention can effectively treat a disease associated with excessive activity by inhibiting a major transcription process using a recombinant fusion protein comprising a DNA binding domain and a protein transport domain (PTD) of a GATA- 3 transcription factor.

1 is a recombinant fusion protein (hereinafter referred to as Hph-1-GT3-DBD) of GATA-3 DNA binding domain (G3D) and Hph-1-PTD used as one embodiment of the present invention.
2 is a chromosome blue photograph of the Hph-1-GT3-DBD recombinant fusion protein used as one embodiment of the invention.
FIG. 3 is a Western blot photograph showing the transfection efficiency and intracellular stability of Hph-1-GT3-DBD recombinant fusion protein used as one embodiment of the invention to cuticular T cells by concentration, time.
FIG. 4 is a photograph showing a Hph-1-GT3-DBD recombinant fusion protein used as an embodiment of the invention transferred to HeLa cells through a confocal microscope.
FIG. 5 is a CCK-8 test result showing cytotoxicity against spleen cells obtained from rats of Hph-1-GT3-DBD recombinant fusion protein used as one embodiment of the invention.
FIG. 6 is a graph showing the transcriptional inhibitory effect of Hph-1-GT3-DBD recombinant fusion protein used as one embodiment of the present invention. In order to examine the transcription inhibition effect of Hph-1-GT3-DBD recombinant fusion protein, wild type GATA-3 and an IL-4 promoter plasmid having luciferase Nucleus injection, and luciferase detection.
Figure 7 is a graph showing that the Hph-1-GT3-DBD recombinant fusion protein used as an embodiment of the invention inhibited the expression of IL-4, IL-13 expressed by spleen cells activated by CD3 and CD28 antibody to be. In addition, IL-13 expression was inhibited by Hph-1-GT3-DBD even in artificially differentiated TH2 cells in vitro.
FIG. 8 shows the results of FACSCalibur and IL-2 ELISA that Hph-1-GT3-DBD recombinant fusion protein used as one embodiment of the invention does not affect T cell activation.
FIG. 9 is a graph showing that the Hph-1-GT3-DBD recombinant fusion protein used as an example of the invention effectively reduces various inflammatory cells in an allergic asthma animal model through cell differentiation staining.
FIG. 10 shows whether the Hph-1-GT3-DBD recombinant fusion protein used as an embodiment of the invention shows a therapeutic effect by reducing secretion of TH2-specific cytokines IL-4 and IL-5 in an allergic asthma animal model It is a graph confirmed by ELISA.
Fig. 11 is a photograph showing hematoxylin & eosin staining of hematoxylin & eosin in terms of histological effect of the Hph-1-GT3-DBD recombinant fusion protein used as an example of the invention in an allergic asthma animal model .

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  1: Preparation of recombinant fusion proteins of transcription factor derivatives and protein transport domains

(B) -Hph-1 protein was prepared by cloning the protein transport domain Hph-1 (SEQ ID NO: 1) into the transcription factor derivative GT3-DBD (SEQ ID NO: 2, DNA binding domain of the transcription factor) 1-GT3-DBD. ≪ / RTI > This recombinant fusion DNA was transformed into the BL21 star (DE3) pLys S E. coli strain. After transformation, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added and the protein was induced to express at 20 ° C. for 20 hours. Since E. coli expressing proteins express endotoxin such as LPS, they were first purified through SP beads to remove them. Cells were disrupted with binding buffer (300 mM NaCl, 50 mM NaH ₂ PO ₄ , pH 7.2) and the cells were disrupted using an ultrasonic disrupter. Then, the supernatant was collected by centrifugation, bound to the SP beads, put into a column, and proteins were separated by elution buffer (2M NaCl, 50 mM NaH ₂ PO ₄ , pH 8.0). Then, to separate pure recombinant fusion proteins, six histidine (Histidine) artificially bound to the front of the protein was used to bind the Ni-NTA beads. Place the lysate to the column containing the protein wash buffer for (30mM _{imidazole, 300mM NaCl, 50mM NaH 2 PO} 4, pH 8.0) fully cleaned, and elution buffer for (1M _{imidazole, 300mM NaCl, 50mM NaH 2 PO} 4 , pH 8.0) to separate the protein from Ni-NTA. Finally, PD-10 was used to remove NaCl and imidazole while changing the buffer to 10% glycerol PBS. The finally obtained protein (SEQ ID NO: 3, recombinant fusion protein) was stored at -80 DEG C until the experiment.

Example  2: Confirmation of intracellular delivery of recombinant fusion protein - Identification of protein transport domain function

<2-1. Western Blot  using Intracellular  Delivery confirmation>

5, 2, 1, 0.5, 0.1, 0.05, 0.01 μM) of the Hph-1-GT3-DBD of Example 1 using the splenic T cells and C57BL / 0, 1, 2, 4, 8, 12, 24, 36 h) and the protein transfer was confirmed by Western blotting. And it was confirmed that the protein was maintained and maintained in the cell culture even after 36 hours. In addition, the stability of the delivered protein in the cells was examined. Most of the cells were transferred to the cells within 1 hour, and gradually decomposed in the cells, and most of them were decomposed in 24 ~ 36 hours. This suggests that the amount of the recombinant fusion protein in the body can be regulated by controlling the amount of protein and the frequency of treatment by causing a small side effect in the cell by decomposing the once delivered protein after a certain period of time.

<2-2. Determine if it is delivered to the nucleus of the cell using antibodies>

2 μM of Hph-1-GT3-DBD recombinant fusion protein of Example 1 was incubated with HeLa cells for 2 hours, washed with PBS, made clear with cells using Triton X-100, Antibodies were conjugated to the recombinant fusion protein. Next, the nuclei of the cells were stained using DAPI stain, and the position of the fluorescence was confirmed through a fluorescence microscope to confirm the location of the recombinant fusion protein. As a result, it was confirmed that the protein was well transferred to the nucleus of the cell by the property of PTD (see FIG. 4).

Example  3: Hph -One- GT3 - DBD  Identification of specific transcription factor inhibitory effects of recombinant fusion proteins

<3-1. Cytokine IL -4 production inhibition>

In order to directly confirm whether the Hph-1-GT3-DBD recombinant fusion protein of Example 1 binds to the promoter of the IL-4 cytokine gene instead of the wild-type GATA-3 and suppresses the expression, a luciferase reporter gene Respectively. First, the HeLa cells were transfected with the IL-4 promoter having the F. luciferase gene and wild-type GATA-3 gene, and then the Hph-1-GT3-DBD recombinant fusion protein was treated. As a result, it was confirmed that the expression of luciferase was effectively inhibited when the recombinant fusion protein was treated. This implies that Hph-1-GT3-DBD acts as a competitive inhibitor of wild-type GATA-3, blocking the binding site of the IL-4 promoter.

<3-2. Cytotoxicity>

Cytotoxicity tests were performed to show that LPS was completely removed from proteins expressed from Escherichia coli strains and showed no toxicity in cells or animals. After delivering various concentrations of the proteins to the splenocytes, WST-8, a substrate for coloring, was added by dihydrogenase present in living cells and cultured together. As a result, regardless of the concentration of the treated protein, it was confirmed that no toxicity was observed compared with the untreated cells.

Example  4: Hph -One- GT3 - DBD  Identification of the specific inhibitory effect of the transcription factor of the recombinant fusion protein: Identification of the function of the transcription factor binding domain

<4-1. Cytokine IL -4, IL - Thirteen  Generation inhibition>

Spleen cells were stimulated with anti-CD3 and CD28 antibodies to confirm cytokine secretion from activated T cells. Splenocytes were isolated from the spleen of 7-week-old female BALB / c mice and Hph-1-GT3-DBD of Example 1 was cultured for 2 hours to deliver the recombinant fusion protein into cells. The cells were plated in 12 wells coated with 1 μg / ml of anti-CD3 antibody together with 0.5 μg / ml of anti-CD28 antibody and cultured for 72 hours. The amount of cytokine was then measured using an ELISA present in the culture. In addition, the expression levels of CD69 and IL-2, which are indicators of T cell activation, were confirmed by FACSCalibur and ELISA, respectively, indicating that they were constantly activated regardless of treatment with Hph-1-GT3-DBD. On the other hand, IL-4 and IL-13, TH2 cell-specific cytokines, were significantly inhibited by Hph-1-GT3-DBD treatment. This confirms that the Hph-1-GT3-DBD recombinant fusion protein specifically acts on TH2 cells (see FIG. 7).

<4-2. TH2 &Lt; RTI ID = 0.0 &gt;

After splenocytes were isolated from the rat of Example 4-1, CD4 ⁺ CD25 ^- CD62L ⁺ immature T cells were isolated, which were not exposed to any antigen, using MACS (Magnetic Cell Sorting). The immature CD4 T cells were incubated with 1 μM and 2 μM recombinant fusion proteins of Hph-1-GT3-DBD for 2 hours. Then, 0.5 μg / ml of anti-CD28 antibody was added to 12 wells coated with 1 μg / ml of anti-CD3 antibody Lt; / RTI &gt; for 72 hours. At this time, IL-4 cytokine and anti-IFN antibody were added together to differentiate into TH2 cells. After 72 hours, the amount of each cytokine was determined by ELISA. As a result, IL-13, a typical cytokine of TH2, was significantly reduced. From these results, it was found that the Hph-1-GT3-DBD recombinant fusion protein can also function in the differentiation into TH2 (see FIG. 7).

Example  5: Allergic  In asthmatic animal models Hph -One- GT3 - DBD  Effect of Recombinant Fusion Protein

<5-1. Allergic  Effects on asthmatic animal models>

Six-week old male BALB / c rats were stimulated by injection of 100 μg of ovalbumin and 20 mg of aluminum hydroxide into the abdominal cavity on days 1 and 14. On the 21st, 22nd, and 23rd day, 75 μg of Hph-1-GT3-DBD protein was injected intranasally into the lungs and bronchi, and local stimulation was given with 150 μg of ovalbumin. On the 25th day, the cells were isolated from the BAL fluid obtained from the rats and the number of inflammatory cells was counted through differential staining. As a result, it was confirmed that the number of inflammatory cells was greatly reduced by treatment with Hph-1-GT3-DBD (see FIG. 9). Meanwhile, splenocytes from mice treated with recombinant fusion proteins were isolated and reactivated with anti-CD3 / CD28 antibody. In this case, since the rat has already been exposed to the antigen once in the body, when the stimulus is given again in vitro, stronger activity occurs and secretes cytokine again. IL-4, IL-5 and IL-13, which are representative cytokines of TH2, were significantly reduced in rats injected with Hph-1-GT3-DBD recombinant fusion protein by ELISA as the amount of cytokine present in the culture medium. Respectively. (See Fig. 10).

<5-2. Allergic  Histological Effects in Asthma Animal Model>

In order to investigate how Hph-1-GT3-DBD changes the morphology and inflammation of the lungs and bronchi using immunohistochemistry, the present inventors examined the lung and bronchus of an animal model in which erythrocytes were removed by PBS washing After storing in formaldehyde for 24 hours, a paraffin tissue section was prepared. This paraffin tissue section was stained with H & E and PAS. As a result, it was confirmed that the tissue of the bronchial tree was restored to the normal state when the Hph-1-GT3-DBD recombinant fusion protein was treated as compared with the disease- I could. In addition, it was confirmed that infiltration of inflammatory cells was also decreased compared with the disease induction model. Based on these results, it was demonstrated that Hph-1-GT3-DBD plays a role in suppressing allergic asthma by reducing the response of TH2 (see FIG. 11).

Example  6: Recombination Of the fusion protein Acute Toxicity Experiment

The acute toxicity test was carried out as described below using specific pathogen-free (SPF) SD rats of 6 weeks old supplied from the experimental supply center.

The recombinant fusion protein of Example 1 of the present invention was orally administered to two animals per each group at a dose of 1 g / kg once, followed by observation of the animal's mortality, clinical symptoms and weight change, and hematology test and blood biochemistry The autopsy was performed, and autopsy was performed to observe whether or not the organs and thoracic organs were abnormal.

As a result of the experiment, there were no clinical symptoms or dead animals in all animals treated with the test substance, and no toxic change was observed in weight change, blood test, blood biochemical test, and autopsy findings. As a result, it was confirmed that the recombinant fusion protein of Example 1 of the present invention did not show any toxicity at 1 g / kg or more in rats, and that the oral LDL was determined to be a safe substance with an LD50 of 1 g / kg or more .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the present invention is not limited to the disclosed exemplary embodiments, but various changes and modifications may be made without departing from the scope of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but are intended to illustrate and not limit the scope of the technical spirit of the present invention. The scope of protection of the present invention should be construed according to the claims, and all technical ideas which are within the scope of the same should be interpreted as being included in the scope of the present invention.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A PHARMACEUTICAL COMPOSITION USING A FUSION PROTEIN COMPRISING          DNA BINDING DOMAIN FROM GATA-3 OF TRANSCRIPTION FACTORS AND          PROTEIN TRANSDUCTION DOMAIN FOR TREATING AN ALLERGIC ASTHMA ANDA          METHOD USING THE SAME <130> IPDC-40046 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 11 <212> PRT <213> Artificial <400> 1 Tyr Ala Arg Val Arg Arg Arg Gly Pro Arg Arg   1 5 10 <210> 2 <211> 108 <212> PRT <213> Artificial <400> 2 Cys Val Asn Cys Gly Ala Thr Ser Thr Pro Leu Trp Arg Arg Asp Gly   1 5 10 15 Thr Gly His Tyr Leu Cys Asn Ala Cys Gly Leu Tyr His Lys Met Asn              20 25 30 Gly Gln Asn Arg Pro Leu Ile Lys Pro Lys Arg Arg Leu Ser Ala Ala          35 40 45 Arg Arg Ala Gly Thr Ser Cys Ala Asn Cys Gln Thr Thr Thr Thr Thr      50 55 60 Leu Trp Arg Arg Asn Ala Asn Gly Asp Pro Val Cys Asn Ala Cys Gly  65 70 75 80 Leu Tyr Tyr Lys Leu His Asn Ile Asn Arg Pro Leu Thr Met Lys Lys                  85 90 95 Glu Gly Ile Gln Thr Arg Asn Arg Lys Met Ser Ser             100 105 <210> 3 <211> 160 <212> PRT <213> Artificial <400> 3 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Gly Tyr   1 5 10 15 Ala Arg Val Arg Arg Arg Gly Pro Arg Arg Gly Gly Ser Tyr Ala Arg              20 25 30 Val Arg Arg Arg Gly Pro Arg Arg Glu Phe Cys Val Asn Cys Gly Ala          35 40 45 Thr Ser Thr Pro Leu Trp Arg Arg Asp Gly Thr Gly His Tyr Leu Cys      50 55 60 Asn Ala Cys Gly Leu Tyr His Lys Met Asn Gly Gln Asn Arg Pro Leu  65 70 75 80 Ile Lys Pro Lys Arg Arg Leu Ser Ala Ala Arg Arg Ala Gly Thr Ser                  85 90 95 Cys Ala Asn Cys Gln Thr Thr Thr Thr Thr Leu Trp Arg Arg Asn Ala             100 105 110 Asn Gly Asp Pro Val Cys Asn Ala Cys Gly Leu Tyr Tyr Lys Leu His         115 120 125 Asn Ile Asn Arg Pro Leu Thr Met Lys Lys Glu Gly Ile Gln Thr Arg     130 135 140 Asn Arg Lys Met Ser Ser Leu Glu Asp Tyr Lys Asp Asp Asp Asp Lys 145 150 155 160

Claims (22)

A fusion protein comprising a DNA binding domain of GATA-3 and Hph-1 which is a protein transport domain, for the treatment or prevention of an autoimmune disease. delete delete delete The method according to claim 1,
Wherein the Hph-1 is composed of SEQ ID NO: &lt; RTI ID = 0.0 &gt; 1. &Lt; / RTI &gt; The method according to claim 1,
Wherein the DNA binding domain of GATA-3 is composed of SEQ ID NO: 2. 2. A pharmaceutical composition for the treatment or prevention of autoimmune diseases, The method according to claim 1,
Wherein the fusion protein is comprised of SEQ ID NO: 3. 3. A pharmaceutical composition for the treatment or prevention of an autoimmune disease, A DNA binding domain of GATA-3 and Hph-1 as a protein transport domain. delete delete delete 9. The method of claim 8,
Lt; RTI ID = 0.0 &gt; 1. &Lt; / RTI &gt; 9. The method of claim 8,
Wherein the DNA binding domain of GATA-3 is comprised of SEQ ID NO: 2. 9. The method of claim 8,
Wherein the fusion protein is comprised of SEQ ID NO: 3. The method according to claim 1,
A pharmaceutical composition for the treatment or prevention of an autoimmune disease, which further comprises a pharmaceutically acceptable excipient. delete delete The method according to claim 1,
Wherein the autoimmune disease is selected from the group consisting of allergic asthma, atopic dermatitis, systemic lupus erythematosus, ulcerative colitis, and pelargatitis. delete delete delete delete

Images