KR20150001223A - A Composition containing Y structured DNA for enhancing Immunity and Anti-Cancer Effect - Google Patents

Abstract

In the present invention, DNA having a Y-structure binds to TLR9 to increase IFN-? And COX2 promoter activity, to increase the secretion of IL-12 and TNF-? In dendritic cells, CD80, and CD86 expression, and the acquired immune response is effectively induced by stimulating innate immune cells. The DNA having the Y-shaped structure according to the present invention is useful as a composition for enhancing an immune effect or a pharmaceutical composition for the prevention or treatment of immune diseases And may be usefully used as a composition for enhancing the anticancer effect which can enhance the efficacy of the anticancer drug by administering it in combination with an anticancer drug.

Description

[0001] The present invention relates to a composition for promoting immunity and an anticancer effect comprising DNA of Y-structure as an active ingredient,

The present invention relates to a composition for enhancing immunity and anticancer effect comprising DNA having Y-shaped structure formed by complementary binding of three oligonucleotides as an active ingredient.

Bacterial DNA has an immunostimulatory effect on activating B cells and natural killer cells, but vertebrate DNA has not been shown to have such an effect. Presently, it is recognized that the immunostimulatory effect of such bacterial DNA is a consequence of the presence of unmethylated CpG dinucleotide under a special base environment (CpG motif), which is commonly found in bacterial DNA, Methylated and underlined. The immunostimulatory effect of bacterial DNA can be mimicked by synthetic oligodeoxynucleotides (ODN) containing these CpG motifs. Since such CpG ODNs have a highly stimulatory effect on human and murine leukocytes, B cell proliferation; Cytokine and immunoglobulin secretion; Natural killer (NK) cell lytic activity and IFN-secretion; And DCs and other antigen presenting cells to secrete Th1-like cytokines that are important in promoting the development of cytokines, particularly Th1-like T cell responses, by expressing co-stimulatory molecules. The immunostimulatory effect of the natural phosphodiester main chain CpG ODN is highly CpG-specific in that the effect is markedly lowered when the CpG motif is methylated, changed to CpG, or removed or changed.

In addition, CpG immunostimulatory oligonucleotides have been reported to enhance the effect of adjuvants in vaccine settings. U.S. Patent No. 6,406,705 B1 describes the use of a combination of CpG oligonucleotides, non-nucleic acid enhancers and antigens to induce antigen-specific immune responses. Non-nucleic acid adjuvants included adjuvants to produce a depot effect, adjuvants to stimulate the immune system, and adjuvants having both of these activities. However, the above patent does not teach or suggest the immunostimulatory effect using non-CpG oligonucleotides.

U.S. Patent No. 6,406,705

It is an object of the present invention to provide a DNA having a novel structure capable of promoting an immune effect and to utilize the DNA as a pharmaceutical composition for the treatment or prevention of an immune disease or an anticancer agent capable of enhancing the anti- As a composition for enhancing the effect.

In order to accomplish the above object, the present invention provides a recombinant vector comprising complementary binding of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or oligonucleotide having a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: And a DNA having a Y-shaped structure formed by complementary binding of the DNA of the present invention as an active ingredient.

In one embodiment of the present invention, the pharmaceutical composition for promoting immune effect comprises a Y-shaped DNA formed by complementary binding of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 And may form a polymer by complementary bonding between DNAs of the Y-type structure.

In one embodiment of the present invention, the DNA of the Y-type structure may induce a congenital immune response.

In one embodiment of the present invention, the congenital immune response may be induced by increasing the promoter activity of IFN-? Or COX-2 in macrophages.

In one embodiment of the present invention, the congenital immune response may be induced by increasing secretion of IL-12 or TNF-a in dendritic cells.

In one embodiment of the present invention, the congenital immune response may be induced by increasing the expression of CD80 and CD86 on the dendritic cell surface.

In one embodiment of the present invention, the Y-shaped DNA may bind to TLR9 to induce a congenital immune response.

In addition, the present invention relates to a complementary binding of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, or a complementary binding of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: Which comprises DNA of the Y-shaped structure formed by the DNA of the present invention as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating an immunological disease.

In one embodiment of the present invention, the immunological disease may be selected from the group consisting of autoimmune diseases, inflammatory diseases, and transplantation rejection diseases of cells, tissues or organs.

In one embodiment of the present invention, the immune disorder is selected from the group consisting of Behcet's disease, multiple myelitis / dermatomyositis, autoimmune hemolytic disease, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Atherosclerosis, neurodegenerative disease, meningitis, Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin dependent diabetes mellitus, eosinophilic epidermolysis epididymitis, glomerulonephritis, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the treatment of ulcerative colitis, It may be selected.

YL-DNA and YS-DNA according to the present invention bind to TLR9 to increase expression of IFN-? And COX2, increase secretion of IL-12, TNF- ?, and dendritic cells CD80, and CD86 expressing cells, thereby inducing acquired immune responses effectively. Thus, when the composition is used as a composition for enhancing immunity or as an adjuvant for an anticancer drug, It may improve the prevention effect.

1 is a schematic diagram showing the structures of YL-DNA and YS-DNA.
Fig. 2 shows the results of luciferase analysis showing the degree of activation of the COX-2 promoter (A) and the IFN-? Promoter (B) by YL-DNA and YS-DNA.
FIG. 3 shows the results of qRT-PCR showing the changes in mRNA expression levels of IL-12 and TNF-? By YL-DNA and YS-DNA treatment in bone marrow-derived dendritic cells.
FIG. 4 shows ELISA results showing changes in the expression levels of IL-12 and TNF-α by YL-DNA and YS-DNA treatment in bone marrow-derived dendritic cells.
FIG. 5 shows the results of qRT-PCR showing the amount of CD86 mRNA expressed as an auxiliary stimulating molecule on the surface of bone marrow-derived dendritic cells by YL-DNA treatment.
Fig. 6 shows FACS results showing the expression levels of CD80 and CD86, which are auxiliary stimulating molecules on the surface of bone marrow-derived dendritic cells by YL-DNA and YS-DNA treatment.
7 shows the result of immunoprecipitation in which YL-DNA is directly bound to TLR9.
FIG. 8 shows the results of qRT-PCR in which the amount of IL-12, TNF-α and IFN-β mRNA expression was changed by YL-DNA and YS-DNA treatment in bone marrow derived dendritic cells isolated from wild type and TLR9 deficient mice .
FIG. 9 shows ELISA results of observing changes in the amount of IL-12 expressed by YL-DNA and YS-DNA treatment in bone marrow-derived dendritic cells isolated from wild-type and TLR9-deficient mice.

The present invention is characterized by providing a composition for promoting an immune effect and a pharmaceutical composition for the prevention or treatment of immune diseases, which comprises DNA having a Y-structure as an active ingredient.

The present inventors have paid attention to Y-type structure DNA to develop a novel immunomodulator for enhancing the immune effect and treating immune diseases. The inventors of the present invention focused on the DNA of the Y-type structure by using oligonucleotides The Y-shaped DNA formed by the complementary binding or the complementary binding of the oligonucleotide having the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 was excellent in inducing congenital immune response, , The DNA of the Y-shaped structure formed by the complementary binding of the oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 was found to form a polymer by the complementary bonding of the DNAs of the Y-type structure The DNA of the Y-shaped structure in which such a polymer is formed is superior to the Y-shaped DNA in the monomorphic form, It has been shown to induce a reaction.

These innate immune responses increase IFN-? And COX-2 promoter activity in macrophage RAW264.7 cells and increase the secretion of IL-12 and TNF-a, the immunocytokines in dendritic cells, And increased expression of CD80, CD86, which is an auxiliary stimulating molecule.

In addition, this congenital immune response was not observed in dendritic cells isolated from TLR9-deficient mice. Thus, the Y-shaped DNA according to the present invention induces the congenital immune response by a TLR9-dependent mechanism In particular, it was confirmed through immunoprecipitation analysis that TLR9 and DNA of Y structure induce congenital immune response through direct binding.

Accordingly, the DNA having the Y-structure according to the present invention may be used as a composition for enhancing an immune effect or a pharmaceutical composition for preventing or treating an immunological disease.

In the present invention, the immunological disease refers to a disease in which components of the mammalian immune system cause, mediate, or contribute to a pathological condition of a mammal. In addition, the stimulation or discontinuation of the immune response may include all diseases that have a compensatory effect on the progress of the disease, and the present invention may include diseases caused by an irritable immune response. Examples of such immune disorders include, but are not limited to, autoimmune diseases; Inflammatory disease; And transplantation rejection diseases of cells, tissues or organs.

In addition, one of the most important properties of all normal individuals is that they do not react negatively to the antigenic material that constitutes the self, while non-self antigens are recognized and reacted to remove I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

However, when problems arise in inducing or maintaining such self-tolerance, an immune response to self-antigen occurs, thereby attacking the tissue of self. The disease caused by this process is called autoimmune disease .

In addition, inflammatory diseases include TNF-α, IL-1 (interleukin-1), IL-1 (interleukin-1 and IL-2) secreted from immune cells such as macrophages by overexpression of the human immune system due to harmful stimuli, Inflammatory substances such as inflammatory cytokines such as IL-1, IL-6, prostagladin, luecotriene or nitric oxide (NO).

On the other hand, in order to successfully transplant the organ, the recipient's immune rejection response to the transplanted cells and organs must be overcome. The main mediator of transplantation immune rejection is T cell, which is expressed in the graft. Major histocompatibility complex (MHC) is recognized by T cell receptor (T cell receptor) A reaction occurs. Immunosuppressive agents have been used to reduce these transplant immune rejection responses. A common goal of these immunosuppressive agents is to inhibit T cell-mediated immune responses to grafts. Recently, regulatory T cells have been used to suppress immune responses, Methods to treat refractory diseases have been attempted.

In the present invention, the autoimmune diseases or inflammatory diseases include but are not limited to autoimmune hepatitis, chronic rheumatoid arthritis, insulin dependent diabetes mellitus, ulcerative colitis, Crohn's disease, multiple sclerosis, autoimmune myocarditis, Scleroderma, severe muscular atrophy, multiple myelitis / dermatomyositis, Hashimoto's disease, autoimmune cytopenias, Sjogren's syndrome, vasculitis syndrome and systemic lupus erythematosus.

In the present invention, " treatment ", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term " treatment ", as used herein, refers to an act of treating when " treating " is defined as above. Thus, "treatment" or "treatment regimen" of an immune disorder in a mammal may include one or more of the following:

(1) inhibiting the growth of the immune system, i.e., inhibiting its development,

(2) preventing the spread of immune diseases, i.e., preventing metastasis,

(3) alleviation of immune diseases.

(4) preventing the recurrence of immune disorders, and

(5) palliating symptoms of immune disorders

The composition for the prevention or treatment of immune diseases according to the present invention may contain a pharmaceutically effective amount of Y-shaped DNA alone or may comprise at least one pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat symptoms of an immune disease.

Also, the pharmaceutically acceptable hereinabove is physiologically acceptable and refers to a composition which, when administered to a human, does not normally cause an allergic reaction such as a gastrointestinal disorder, dizziness, or the like. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing or treating immunological diseases according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the dose of the active ingredient may be appropriately determined depending on the route of administration, The composition of the present invention can be appropriately selected according to various factors such as body weight and the severity of the patient and the composition for preventing or treating immune diseases according to the present invention can be used in combination with known compounds having the effect of preventing, Lt; / RTI >

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Values obtained in the following examples are expressed as mean + - SD.

≪ Example 1 >

YL - DNA Wow YS - DNA Production

The three oligonucleotides described in the following table were dissolved in the same amount in annealing buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 50 mM NaCl) and sterilized Milli-Q water was added to give a final concentration of 0.5 mM The mixture was reacted at 95 for 5 minutes, 65 for 2 minutes, 62 for 1 minute, and slowly lowered to 4. YL-DNA and YS-DNA were electrophoresed at 200 V for 1.5-2 hours using 21% Respectively.

Base sequence
YL-DNA
Y01: 5'-ACG TTG GAT CCG CAT GAC ATT CGC CGT AAG-3 '(SEQ ID NO: 1) Y02: 5'-ACG TCT TAC GGC GAA TGA CCG AAT CAG CCT-3 '(SEQ ID NO: 2) Y03: 5'-ACG TAG GCT GAT TCG GTT CAT GCG GAT CCA-3 '(SEQ ID NO: 3)
YS-DNA
Y01: 5'-TGA CTG GAT CCG CAT GAC ATT CGC CGT AAG-3 '(SEQ ID NO: 4) Y02: 5'-TGA CCT TAC GGC GAA TGA CCG AAT CAG CCT-3 '(SEQ ID NO: 5) Y03: 5'-TGA CAG GCT GAT TCG GTT CAT GCG GAT CCA-3 '(SEQ ID NO: 6)

As can be seen from the above table, the YL-DNA has an ACGT nucleotide sequence complementary to Y01, Y02 and Y03, but YS-DNA does not have this nucleotide sequence.

Therefore, as shown in FIG. 1, YL-DNA may exist as a condensed complex polymer in which monomers are bonded to each other, and in the case of YS-DNA, a complementary binding end is lacking And may exist only in the form of a monomer.

< Example  2>

YL - DNA Wow YS - DNA of IFN -β and COX -2 promoter activation effect analysis

Some oligonucleotides such as CpG DNA are known to effectively induce an immune response, and the present inventors have tried to confirm whether YL-DNA and YS-DNA can induce such an immune response.

For this purpose, a cell line was constructed by permanently expressing IFN-? Promoter luciferase or COX-2 promoter luciferase reporter gene in RAW264.7 cells. Cells were treated with 0.01, 0.1 and 1 μM of YL-DNA and YS-DNA, respectively, and the cells were lysed using luciferase dissolution buffer for 8 hours. Then, luciferase assay kit (promega, USA ), The promoter activity of IFN-β and COX-2 was observed according to the manufacturer's method.

As a result, as shown in Fig. 2, it was confirmed that IFN-? And COX-2 promoter activity was increased in a concentration-dependent manner by YL-DNA and YS-DNA treatment. On the other hand, YL-DNA showed better IFN-? And COX-2 promoter activity than YS-DNA.

&Lt; Example 3 >

YL - DNA Wow YS - DNA Of immunocytochemicals

The inventors of the present invention confirmed that YL-DNA and YS-DNA increase the IFN-? And COX-2 promoter activities of macrophages and induce congenital immune responses through Example 2 above. Thus, the present inventors have determined whether YL-DNA and YS-DNA induce secretion of immunocytokines from dendritic cells known to be important for acquired immune cell activation.

For this purpose, YL-DNA and YS-DNA according to the present invention were treated with 0.01, 0.1, and 1 μM of dendritic cells, respectively, and the amount of IL-12 and TNF-α mRNA was measured by quantitative RT- Respectively. The total RNA was separated from the bone marrow-derived dendritic cells treated with YL-DNA and YS-DNA into Welprep ^™ reagent (Jeil Biotechservices Inc., Daegu, Korea) and the reverse transcription reaction was performed using ImProm II ^™ reverse transcription reagent (Promega, Madison, (Bio-Rad) using 1 μl of cDNA as a template using IQ ^™ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) according to the manufacturer's protocol . Quantification was derived by the efficiency-corrected Cq method. Relative expression levels were normalized using β-actin.

As a result, as shown in FIG. 3, the amount of mRNA of IL-12 and TNF-α was increased in a concentration-dependent manner by treatment with YL-DNA and YS-DNA, MRNA levels of IL-12 and TNF-α were higher than those of IL-12 and TNF-α expressed by YS-DNA treatment.

The present inventors further examined the secretion amount of IL-12 and TNF-α protein. For this purpose, YL-DNA and YS-DNA were treated with 0.01, 0.1 and 1 μM of bone marrow-derived dendritic cells, respectively, And the amount of IL-12 and TNF-a secreted by the mice were measured.

As a result, the amount of protein secretion was similar to the amount of mRNA expression, and the amount of IL-12 and TNF-α secretion was further increased in YL-DNA treatment than in YS-DNA treatment (see FIG. 4).

These results suggest that YL-DNA and YS-DNA induce secretion of IL-12 and TNF-α in dendritic cells and contribute to acquired immune cell activation.

<Example 4>

YL - DNA Wow YS - DNA Auxiliary stimulation by co - stimulatory ) Molecular expression analysis

In order to observe the effect of YL-DNA and YS-DNA on the immune cell activity, we tried to measure the expression of co-stimulating molecules. In other words, it is known that ancillary stimulating molecules such as CD80 and CD86 are expressed on the surface of activated innate immune cells, activating acquired immune cells. As a result, YL-DNA and YS- And CD86 expression.

To this end, dendritic cells were first treated with YL-DNA (0.01, 0.1, 1 [mu] M) for 4 hours, and the expression level of CD86 mRNA was examined using qRT-PCR in the same manner as described in Example 3 above. As a result, it was confirmed that the expression level of CD86 mRNA was increased depending on the YL-DNA treatment concentration (see FIG. 5).

Meanwhile, the present inventors analyzed CD80 and CD86 expression on the surface of dendritic cells using a flow cytometer. That is, dendritic cells were treated with YL-DNA or YS-DNA (0.01, 0.1, 1 μM) CD80 and CD86 expression were analyzed by flow cytometry.

As a result, it was confirmed that the expression of CD80 and CD86 on the surface of dendritic cells was increased in a concentration-dependent manner by treatment with YL-DNA and YS-DNA. This expression pattern was found to be more conspicuous especially when treated with YL-DNA (see FIG. 6).

< Example  5>

YL - DNA Wow TLR9 Whether or not it is combined

TLR9 is known to directly bind to unmethylated DNA (CpG DNA) contained in bacteria or viruses to induce an immune response. The inventors of the present invention tried to confirm whether YL-DNA binds directly to TLR9.

For this, plasmids for expression of mTLR9-Myc / His were transfected into HEK293T cells using SuperFect Transfection Reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol and cultured for 24 hours. The lysate was reacted with biotinylated YL-DNA for 4 hours, followed by additional reaction with NA-bead for 2 hours, immunoprecipitated and immunoblotted with anti-Myc antibody.

As a result, as shown in FIG. 7, it was confirmed that TLR9 and YL-DNA were directly coupled.

< Example  6>

In primary cultured dendritic cells TLR9  Expression of Immunocytochemical Expression

The present inventors confirmed that YL-DNA binds directly to TLR9 through the above Example 5. Further experiments were conducted using dendritic cells derived from bone marrow of TLR9 deficient mice to clarify its mechanism of action. For this purpose, YL-DNA and YS-DNA were treated with dendritic cells derived from wild-type and TLR9-deficient mice, and IL-12, TNF-α and IFN-β levels were compared.

YL-DNA (1 μM), YS-DNA (1 μM), negative control TLR7 ligand R848 (100 ng / ml), and positive control group, respectively, were used for the primary culture of dendritic cells derived from TLR9 deficient mice and wild- IL-12, TNF-α, and IFN-β mRNA expression levels and IL-12 protein expression levels were measured by qRT-PCR and ELISA assay after treatment with CpG1668 (1 μM).

As a result, as shown in FIGS. 8 and 9, the amount of IL-12, TNF-α, and IFN-β mRNA was increased by YL-DNA and YS-DNA and the amount of protein expressed by IL-12 was increased.

Through the above-described embodiment, DNA having a Y-type structure can activate dendritic cells, which are the major antigen-presenting cells of the innate immune response, to induce the production of immunocytokines and auxiliary stimulating molecules, thereby inducing acquired immune responses . Accordingly, the DNA having the Y-structure according to the present invention may be useful as an adjuvant for chemotherapeutic agents capable of supporting various immune enhancers or anticancer treatments for various immune diseases.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> A Composition containing Y structured DNA for enhancing Immunity          and Anti-Cancer Effect <130> PN1303-115 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> one of the three polynucleotides for forming self-assemblable Y          structured DNA <400> 1 acgttggatc cgcatgacat tcgccgtaag 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> one of the three polynucleotides for forming self-assemblable Y          structured DNA <400> 2 acgtcttacg gcgaatgacc gaatcagcct 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> one of the three polynucleotides for forming self-assemblable Y          structured DNA <400> 3 acgtaggctg attcggttca tgcggatcca 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> one of the three polynucleotides for forming non-self-assemblable Y          structured DNA <400> 4 tgactggatc cgcatgacat tcgccgtaag 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> one of the three polynucleotides for forming non-self-assemblable Y          structured DNA <400> 5 tgaccttacg gcgaatgacc gaatcagcct 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> one of the three polynucleotides for forming non-self-assemblable Y          structured DNA <400> 6 tgacaggctg attcggttca tgcggatcca 30

Claims (10)

The complementary binding of the oligonucleotide having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or the complementary binding of the oligonucleotide having the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: Wherein the composition comprises DNA of the structure as an active ingredient. The method according to claim 1,
The DNA of the Y-shaped structure formed by the complementary binding of the oligonucleotide having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 forms a polymer by complementary binding of DNAs of the Y- Wherein the immunological effect enhancing pharmaceutical composition is a composition for enhancing immunity. The method according to claim 1,
Wherein the DNA of the Y-type structure induces a congenital immune response. The method of claim 3,
Wherein said congenital immune response is induced by increasing the promoter activity of IFN-? Or COX-2 in macrophages. The method of claim 3,
Wherein said congenital immune response is induced by increasing the secretion of IL-12 or TNF-a in dendritic cells. The method of claim 3,
Wherein said congenital immune response is induced by increasing the expression of CD80 and CD86 on the dendritic cell surface. The method of claim 3,
Wherein the Y-shaped DNA binds to TLR9 to induce a congenital immune response. The complementary binding of the oligonucleotide having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 or the complementary binding of the oligonucleotide having the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: Wherein the composition comprises DNA as an active ingredient. 9. The method of claim 8,
Wherein said immune disease is selected from the group consisting of autoimmune diseases, inflammatory diseases, and transplantation rejection diseases of cells, tissues or organs. 9. The method of claim 8,
The immunological diseases are selected from the group consisting of Behcet's disease, multiple myelitis / dermatomyositis, autoimmune hemocytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Goodpitcher's syndrome, autoimmune meningitis, Sjogren's syndrome, , Addison's disease, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, eosinophilic epidermolysis, epididymitis, glomerulonephritis, Graves' disease, Hiramoto disease, hemolytic anemia, Prevention of immune disorders characterized by being selected from multiple sclerosis, myasthenia gravis, pemphigoid, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, mucinous anemia and ulcerative colitis Or a pharmaceutical composition for therapeutic use.

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