KR100924085B1 - Oligonucleotides derived from mycobacterium for stimulating immune function, treating immune-related diseases, atopic dermatitis and/or protecting normal immune cell - Google Patents

Abstract

Oligonucleotides for immune response control are disclosed. This oligonucleotide has effects that can be used to enhance immune function, treat various immune diseases and treat atopic skin diseases, or to survive normal immune cells.

Description

OLIGONUCLEOTIDES DERIVED FROM MYCOBACTERIUM FOR STIMULATING IMMUNE FUNCTION, TREATING IMMUNE-RELATED DISEASES, ATOPIC DERMUNETING NORMOR CELL}

The present invention can be used to enhance the immune response and maintain the balance of the immune response, and relates to a therapeutic use, which is effective for various immune diseases such as atopic dermatitis, oligonucleotides for controlling immune response from Mycobacterium. In more detail, the oligonucleotide having three CpG motifs involved in boosting the immune response, the effects of sequencing modifications, and the immunoadjuvant by the phosphodiester type oligo, Th1 / It is an invention that can be used for the treatment of various immune diseases and atopic dermatitis by balancing Th2 immune response, and the effect of increasing the survival of cells to irradiation.

The immune system, which begins through the innate immune system, must be balanced and finely regulated. In other words, precise regulation of immunity and tolerance, balance of T helper type 1 (Th1) and T helper type 2 (Th2) immunity, inflammation and unresponsiveness is essential. However, unfortunately, many immune disease therapies developed so far have not been properly controlled in the immune system, resulting in problems such as autoimmune diseases, allergic diseases, and chronic inflammation. However, the innate immune system has a mechanism by which immune cells recognize and recognize pathogen-associated molecular patterns (PAMPs) in response to pathogen invasion. Therefore, there is a need for a strategy for developing an immune disease treatment that minimizes the Evil mechanism by accurately understanding the Good & Evil mechanism using the innate immune system.

In the 1890s, William B. Coley observed the peculiar phenomenon that infection of pathogenic microorganisms could induce anti-cancer effects in cancer patients, and applied bacterial therapy to about 900 cancer patients and confirmed about 40% efficacy. there was. In the 1980s, Japanese researchers recognized the usefulness of Coley's toxin in a new light and demonstrated anticancer effects through the active fraction of Bacillus Calmette-Guerin (BCG), and concluded that the anticancer activity of BCG was derived from the characteristics of its DNA. It was. In 1995, researchers at Kreig et al. Demonstrated that oligodeoxynucleotides (ODNs) of specific base sequences consisting of unmethylated cytosine and guanine induce immune cell activation in the study of antisense oligonucleotides for B cell gene inhibition. In Kreig's view, the anticancer effect of BCG by Japanese researchers is due to the characteristics of BCG DNA, which is not methylated. A new paradigm has been proposed to distinguish non-self DNA.

Early studies on immune activation and regulation of bacteria focused on protein antigens, such as Coley's toxin, which induce antibody production. However, several publications have reported the presence of more potent immunoactivating derivatives in microbial components, demonstrating that the DNA of bacteria can be biased not only by potent immunoactivation but also by specific immunoreactivity for each antigen (6 , 7). The key to immune activation and regulation is the CpG dinucleotide, which consists of two nucleic acid sequences, cytosine and guanine. Recent studies have shown that bacterial DNA also plays a role in activating immune cells that are distinguished from their DNA by vertebrates. Such CpG motifs are very common in bacteria, but not in mammals. Oligo-deoxy nucleotides (CpG-oligodeoxynucleotide, CpG-ODN ) containing CpG motifs and activate the host's defense mechanisms leading to innate (innate) immune response obtained in an immune response (acquired immune responses) (Akdis, CA. Curr Opin Immunol. , 12: 641-646, 2000).

Recently, in order to increase the usefulness of the CpG ODN, a CpG ODN with a modified backbone has been developed. The CpG ODN of the phosphodiester backbone, the basic backbone of DNA, is sensitive to nucleases and degrades rapidly in the body. Therefore, there is a small risk of causing toxicity in vivo. However, its activity is known to be lower than that of CpG ODN of other skeletons (Kwon, HJ. Et al. , Biochem. Biophys. Res. Commun., 311: 129-138, 2003). In contrast, the CpG ODN of the phosphorothioate backbone is artificially altered in structure so that it is not degraded by nucleases in the body. The CpG ODN of the phosphorothioate backbone is more stable in the body and more capable of inducing B cells than the CpG ODN of the phosphodiester backbone. Therefore, CpG ODN modified with phosphorothioate backbone is currently the most widely used. However, the CpG ODN of these phosphorothioate backbones increases the binding of nonspecific ODNs to many proteins and causes toxicity because they are not readily degraded in the body. In addition, CpG ODN of the phosphorothioate backbone exacerbates the incidence and symptoms of arthritis (Deng GM et al., Arthritis & Rheumatisum , 43 (2): 356-364, 2000), and systemic lupus erythematosis (SLE). It has been reported to cause autoimmune diseases such as (Tanaka, T. ei al., J Exp. Med. 175: 597-607, 1992).

Since this century, various substances have been added to vaccines as adjuvants, and these preparations have been designed to maximize the effectiveness of vaccines. At present, however, aluminum salts (alum, Al ₂ O ₃ ) are the only adjuvant approved for use in livestock vaccines, as well as in human vaccines. In a recent study, injection of recombinant hepatitis surface antigen into mice with a combination of alum and CpG-ODN showed a much higher vaccine effect than when only alum was used as an adjuvant (Davis H L. et al., J.) . Immunol. 160: 870-876, 1998). Alum induces a Th2 immune response, resulting in weak cell mediated immunity. On the other hand, CpG ODN was shown to induce the expression of Th1 cytokines strongly induced humoral and cell mediated immune response. However, the CpG-ODN used at this time may cause side effects using the phosphorothioate skeleton.

On the other hand, skin disease refers to all abnormalities appearing on the skin of animals including humans. Among them, atopic dermatitis is a chronic / inflammatory skin disease, which is characterized by severe pruritus, dry skin and eczema skin lesions (Rudikoff, D. et al., Lancet. 351: 1715-1721, 1998). Atopic dermatitis generally tends to be hereditary and is often accompanied by allergic asthma, allergic rhinitis, allergic conjunctivitis and urticaria. A series of immunological abnormalities reported in patients with atopic dermatitis include increased I gE production, decreased number and decreased function of CD8 + suppressor / cytotoxic T lymphocytes, and Th1 ( T −) secreting IFN-gamma. cell Helper type 1) Lymphocyte count. In addition, atopic dermatitis has histologically increased T lymphocytes, monocytes / macrophages with a CD4 + phenotype, mast cells and eosinophils in skin lesions, dendritic cells (DCs) and epidermal Langerhans cells, which are associated with atopic dermatitis. Increased in skin lesions (Imokawa, G., et al., J. Invest. Dermatol., 96: 523-526, 1991).

Many researchers have improved the methods of treating cancer by killing cancer cells using X-rays. However, since radiation is also irradiated with cancer cells surrounding cancer cells, inevitably damage immune cells, thereby inducing a decrease in immune function. B cells by irradiation (Ashwell JD et al., J. Immunol. 136: 3649-3656, 1986), T cells (Prosser JS Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 30 : 459-465, 1976) and macrophages (Yoshihisa K et al., J. Radiat Res. 45: 205-211, 2004) have reported reports of apoptosis (apoptosis). Therefore, there is a need for a method of maintaining the normal immune response by surviving normal immune cells other than cancer cells in radiation therapy for the treatment of diseases such as cancer.

The present invention can be used for the treatment of various immune diseases through adjuvant and balance of immune responses, and is effective in atopic dermatitis and increases the survival of cells against irradiation. In the present invention, Mycobacterium bovis is an invention for oligonucleotides for controlling the immune response derived from BCG.

Accordingly, the present invention provides a CpG oligodeoxynucleotide isolated from Mycobacterium bovis BCG consisting of a nucleotide sequence having at least two or more unmethylated CpG motifs, and an immune response enhancer (immune adjuvant), various immune diseases. The present invention provides a balance of Th1 / Th2 immune response for treatment, an effect that can be used to survive normal immune cells during radiation treatment of intractable diseases such as cancer, and a method for treating or preventing skin diseases.

[Formula]: HKCGTTCRTGTCSGM (SEQ ID NO: 1)

Wherein R is A or G and S is C or G, H is A, T or C, K is G or T and M is C or A.

In the present invention, the oligonucleotide preferably further includes five nucleotides represented by the following general formulas on the 5 'side and the 3' side.

[Formula]: DKMHKCGTTCRTGTCSGMYK (SEQ ID NO: 2)

Wherein R is A or G and S is C or G, H is A, T or C, K is G or T, D is A, G or T, M is C or A, and M is C or A and Y is C or T.

In the present invention, the 'CpG motif' is also referred to as an 'unmethylated cytosine-guanine dinucleotide' ('unmethylated cytosine-phosphate-guanine dinucleotide') linked by a phosphate bond. Refers to a base sequence that contains and activates the immune response. In addition, 'CpG oligodeoxynucleotide (hereinafter referred to as' CpG ODN ')' refers to an oligodeoxynucleotide including at least two or more CpG motifs.

In addition, in the present invention, 'subject' refers to an animal including a mammal, particularly a human. The subject may be a patient in need of treatment.

In the present invention, the oligonucleotide is 5'-AGCAGCGTTCGTGTCGGCCT-3 '(SEQ ID NO: 3), 5'-AGCAGCGTTCGTGTGCGCCT-3' (SEQ ID NO: 4), 5'-AGCAGCGTTCATGTCGGCCT-3 '(SEQ ID NO: 5), Most preferred are 5'-AGCAGCGTTCGTGTCCGCCT-3 '(SEQ ID NO: 6), 5'-GTATTCGTTCGTGTCGTCCT-3' (SEQ ID NO: 7), 5'-TGACTCGTTCGTGTCGCATG-3 '(SEQ ID NO: 8).

MB-ODN of the present invention may be derived from natural sources (eg, chromosomal DNA of M. bovis BCG) and may be chemically synthesized or recombinantly produced. MB-ODN of the present invention can be synthesized using various nucleic acid synthesis techniques and machines known in the art (Ausubel et al., Current Protocols in Molecular Biology, Chs 2. and 4 (Wiley Interscience, 1989; Maniatis, et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., New York, 1982); and US Pat. No. 4,458,066).

MB-ODN of the present invention preferably has a phosphodiester skeleton. The phosphodiester backbone, which is the basic backbone of DNA, is easily degraded by nucleases in vivo and therefore has a low risk of inducing toxicity. MB-ODN of the present invention, unlike the other phosphodiester backbone, is characterized by excellent immune activity in vitro as well as in vivo, unlike other CpG ODN. In addition, the MB-ODN of the present invention may have a modified backbone. It has been known that modification of the oligonucleotide backbone can enhance the activity and / or stability of CpG ODN when administered in vivo. Preferred modifications of the backbone in the MB-ODN of the present invention include phosphorothioate modifications that render it resistant to degradation. Phosphorothioate modifications can occur at the ends, such as the last two or three 5 'or 3' nucleotides can be linked by phosphorothioate bonds. In addition, the MB-ODN of the present invention may be modified to include secondary structures (eg, stem loop structures) that are resistant to degradation. Preferably it may be modified to have one or more partially phosphorothioate modified backbones. Phosphorothioates can be synthesized by automated techniques using phosphoramidate or H-phosphonate chemistry (SE Beaucage et al., Tetrahedron Lett ., 22: 1859, 1981; Froehler et al., Nucl.Acid.Res . , 14: 5399-5407). Another variant, aryl- and alkyl-phosphonates, can be prepared, for example, as described in US Pat. No. 4,469,863, and alkylphosphotriesters (with charged oxygen moieties US Pat. No. 5,023,243 and European Patent 092,574). Alkylated as described herein) can be prepared by automated solid phase synthesis using commercially available reagents. Another modification that makes it less susceptible to degradation is the inclusion of atypical bases such as inosine and quecine as well as acetyl-, thio- and similar modifications of adenine, cytosine, guanine, thymine and uridine. CpG ODNs containing terminally diols such as tetraethylglycol or hexaethyleneglycol are also more resistant to degradation. Others include combinations of phosphodiester and phosphorothioate, phosphorus esters, phosphorates, methylphosphonates, methylphosphorothionates, phosphorodithiate and combinations thereof (Khorana et al., J Molec. Biol., 72: 209, 1972; Goodchild, J. Bioconjugate Chem ., 4: 165, 1990). Such skeletal-modified CpG ODNs have enhanced nuclease resistance, increased cellular uptake, increased protein uptake and / or altered intracellular localization. Intracellular localization can be used to show stronger immunity.

Preferred skeletons of MB-ODNs of the present invention are phosphodiester (hereinafter referred to as 'type O') or phosphorothioate (hereinafter referred to as 'type S') skeletons, the most preferred skeleton being readily degraded in vivo It is O type skeleton which does not cause side effect.

MB-ODN according to the present invention has been shown to induce the humoral immune response by inducing the expression of Th1 cytokines and has an adjuvant activity that can enhance the effect of the vaccine. Specific physiological activities are as follows.

1) Increase production of IL-12 in mouse and mouse spleen immune cells.

2) Activate dendritic cells to induce the expression of IL-12.

3) When HEL was used as an antigen and MB-ODN as an adjuvant, the production of antibody was greatly increased. As a result of the Th1 immune response, IgG2a production was higher than when using CFA.

MB-ODN according to the present invention through the above activity exhibits the effect of enhancing the efficacy of the vaccine. Unlike the known CpG ODN, the MB-ODN of the present invention is characterized by showing almost the same activity regardless of the shape of the skeleton. In the present invention, it was found that the CpG ODN of the present invention modified with the O-type backbone exhibited almost the same activity as when the Sp-type backbone was modified. In addition, it can be effectively used as an adjuvant of the vaccine by inducing the expression of Th1 cytokines to strongly induce a humoral immune response.

MB-ODN according to the present invention regulates the balance of Th1 / Th2 immune responses by inhibiting Th2 cytokines (eg IL-4) and / or inducing Th1 cytokines (eg IL-12). Have physiological activity. Specific physiological activities are as follows. 1) activates the promoter of IL-12 by activating macrophages; 2) activate dendritic cells to induce expression of IL-12; 3) increase production of IL-12 in mice; 4) increases production of IL-12 in mouse spleen immune cells; 5) inhibits expression of cytokines (IL-4 and IL-10) mediated by Th2-lymphocytes; 6) reduce the cell number of CD4 + and CD8 + lymphocytes at the site of atopic dermatitis lesions; 7) reduce serum IgE levels.

MB-ODN according to the present invention through the above activity shows the effect of the treatment or improvement of symptoms of skin diseases. Unlike the known CpG ODN, the CpG ODN of the present invention is characterized by showing almost the same activity regardless of the shape of the skeleton. In the present invention, it was found that the CpG ODN of the present invention modified with the O-type backbone exhibited the same or better activity than that with the S-type backbone. Therefore, all of the MB-ODN of the present invention can be effectively used for the treatment or prevention of skin diseases. In addition, since the expression of Th1 cytokines is maintained to balance the Th1 / Th2 immune response, it can be effectively applied as a therapeutic agent for immune diseases (eg, asthma) caused by the imbalance of the Th1 / Th2 immune response.

MB-ODN according to the present invention has the function of increasing the survival of immune cells. MB-ODN stimulates macrophages and increases the expression of Bcl-xs / L, which inhibits cell death by irradiation. MB-ODN also has the effect of inhibiting cell death by irradiation of B cells. Therefore, MB-ODN can be effectively applied to normalize immune function by increasing normal immune cell survival rate in the treatment of refractory diseases such as cancer by radiation. Specific physiological activities of MB-ODN are as follows: 1) increase the expression of Bcl-xs / L in macrophages; 2) increase the survival rate of macrophages by irradiation; 3) increase the survival rate of B cells by irradiation.

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate the presently preferred embodiments of the invention and, together with the description of the following preferred embodiments, serve to explain the principles of the invention.

Figure 1 was analyzed the chromosomal DNA sequence of E. coli and M. bovis BCG using a computer program. As a result of analysis of all CpG motifs with two bases on both sides of the CG dinucleotide, we found that much more CpG motifs exist in the chromosomal DNA of M. bovis BCG.

Figure 2 shows the results of analyzing the nucleotide sequences in which three CpG motifs are present on 20 base pairs among the nucleotide sequences present in chromosomal DNA of M. bovis BCG. Analysis of 4 and 5 nucleotide sequences between C and C (-CGXXCGXXXCG-, MB-ODN 4/5) and 5 nucleotide sequences between C and C, respectively ( -CGXXXCGXXXCG-, MB-ODN 5/5). 395 oligonucleotides of -CGXXCGXXXCG- form and 354 oligonucleotides of -CGXXXCGXXXCG- form in chromosomal DNA of M. bovis BCG.

Figure 3 was selected and synthesized 71 candidate oligonucleotides for immune response control was used to search for candidates.

Figure 4 shows the IL-8 and IL-12 promoter activation in RAW 264.7 cells treated with 71 oligonucleotides synthesized in Figure 3 in the form of Phosphodiester binding. Figure 4a is a diagram showing the results of comparing the degree of IL-8 promoter activation of macrophages synthesized 35 oligonucleotides of MB-ODN 4/5 form, Figure 4b is MB-ODN 4/5 form Figure 35 shows the synthesis of the oligonucleotide synthesized compared with the degree of IL-12 promoter activation of macrophages, Figure 4c shows the synthesis of 35 oligonucleotides of the MB-ODN 5/5 form macrophages This figure compares the degree of IL-8 promoter activation.

FIG. 5 shows 17 nucleotide sequences of 5 'side and 3' side of CGTTCGTGTCG core of MB-ODN 4/5 # 31 on 20 base pairs among nucleotide sequences present in chromosomal DNA of M. bovis BCG. By selecting the nucleotide sequence (Fig. 5a), the oligonucleotide of the phosphodiester backbone was synthesized to compare the degree of IL-8 promoter activation of macrophages (Fig. 5b).

FIG. 6 shows MB-ODN 4/5 # 31 (M) which reduced the nucleotide sequence of MB-ODN 4/5 # 31 to 15, # 31-CG where CG sequence was replaced with GC sequence, and G of A, T , Or # 31-A, B, C, D oligonucleotides each substituted with C (Fig. 6a) is a diagram showing the results of comparing the degree of IL-8 promoter activation of macrophages (Fig. 6b).

Figure 7 is a comparison of the effects of the backbone of MB-ODN 4/5 # 31 and # 31.14 in the form of phosphodiester and phosphorothioate to the activation of IL-8 and IL-12 promoter in RAW 264.7 cells, a mouse macrophage. 7a and 7b show the synthesis of the backbone of MB-ODN 4/5 # 31 in the form of phosphodiester and phosphorothioate to activate IL-8 promoter in a concentration dependent manner. 7c and 7d show the effect of the backbone of MB-ODN 4/5 # 31 and # 31.14 synthesized in the form of phosphodiester and phosphorothioate on IL-8 and IL-12 promoter activation.

Figure 8 shows the activation of NF-κB when stimulated with RAW 264.7 cell line phosphodiester and phosphorothioate backbone forms of MB-ODN 4/5 # 31. Figure a) shows the location of NF-κB by treating RAW 264.7 cells with MB-ODN 4/5 # 31 (10 μg / ml) for 30 minutes and then immobilizing the cells and performing indirect immunofluorescence with NF-κB p65-specific antisera. Check the figure. Figure b) Electrophoretic Mobility Shift to identify NF-κB that binds to NF-κB consensus binding site after 30 minutes treatment of RAW 264.7 cells with MB-ODN 4/5 # 31 (10 μg / ml). Assay (EMSA) picture.

Figure 9 shows the humoral response of Balb / c mice intraperitoneally immunized with hen egg lysozyme (HEL) by synthesizing the backbone of MB-ODN 4/5 # 31 in the form of phosphodiester and phosphorothioate.

10 is a result of comparing the effect of MB-ODN 4/5 # 31 of the present invention on the expression of IL-12 in dendritic cells compared with the conventional 1826 CpG ODN and non-CpG ODN (2041).

11 is a result of comparing the effects of the skeletal modification of MB-ODN 4/5 # 31 according to the present invention in the production of IL-12 p40 compared with the conventional 1826 CpG ODN and non-CpG ODN (2041). Figure a) shows the production of IL-12 p40 in serum after intraperitoneal immunization of MB-ODN 4/5 # 31 in Balb / c mice. Figure b) shows the production of IL-12 p40 when isolated splenic immune cells from Balb / c mice were treated with MB-ODN 4/5 # 31.

12 is a result confirming by using an animal model that atopic dermatitis is treated by administration of type O MB-ODN 4/5 # 31 according to the present invention.

a: photographs visually observed on day 5 and 7 after application of type O MB-ODN 4/5 # 31 of the present invention to atopic dermatitis lesions on the back of NC / Nga mice; b: Photograph taken by H & E staining after skin type O-type MB-ODN 4/5 # 31 was applied to the back skin of NC / Nga mouse with atopic dermatitis. In the figure, "↔" indicates acanthosis and "→" indicates hyperkeratosis.

Figure 13 is a histochemical analysis of the expression of cytokines (IL-4 and IFN-gamma) in the dorsal skin of NC / Nga mice administered O-type MB-ODN 4/5 # 31 of the present invention, arrows Indicates expression of cytokines.

Fig. 14 shows the results of histochemical analysis of cell numbers of CD4 + and CD8 + lymphocytes in the dorsal skin of NC / Nga mice to which the type O MB-ODN 4/5 # 31 of the present invention was administered.

FIG. 15 shows the results of IgE levels in serum of NC / Nga mice administered with O-type MB-ODN 4/5 # 31 of the present invention. AD shows untreated group.

Figure 16 shows that the expression of Bcl-xs / L is increased when O-type MB-ODN 4/5 # 31 in macrophage RAW264.7 cell line.

Figure 17 is confirmed by MTT assay that the survival rate of RAW264.7 cells increased when the irradiation after pre-treatment MB-ODN 4/5 # 31 to RAW264.7 cells.

Figure 18 shows the flow cytometry after PI staining that the survival rate of RPMI 8226 cells increased when pre-treated with MB-ODN 4/5 # 31 to RPMI 8226 cells, B cell line.

19 is a flow cytometry figure after Annexin V staining shows that the survival rate of RPMI 8226 cells increased when pre-treated with MB-ODN 4/5 # 31 in RPMI 8226 cells, which are B cell lines.

Best Mode for Carrying Out the Invention

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited thereto.

<Example 1>

E. coli  Wow M. bovis  Chromomal DNA sequencing of BCG

<1-1> CpG motif sequencing of chromosomal DNA of E. coli and M. bovis BCG

We analyzed the chromosomal DNA sequences of E. coli and M. bovis BCG using a computer program. The frequency of base sequences consisting of 6 nucleotides in chromosomal DNA of E. coli and M. bovis BCG was calculated using a computer program. The probability of nucleotide sequence of XXCGXX is theoretically 1/4 ⁶ , but the probability of XXCGXX is much higher in chromosomal DNA of E. coli and M. bovis BCG. In addition, it was confirmed that XXCGXX appeared more frequently than E. coli in chromosomal DNA of M. bovis BCG (FIG. 1).

<1-2> M. bovis  CpG ODN Sequencing from BCG's Chromomal DNA

When 20 nucleotide sequences of M. bovis BCG chromosomal DNA were randomly selected, three XXCGXX sequences were selected. Example) GA CG TTGAGT CG TTAA CG AG

Analysis of 4 and 5 nucleotide sequence intervals between C and C (-CGXXCGXXXCG-, MB-ODN 4/5, FIG. 2A ) and 5 nucleotide sequence intervals between C and C As a result (-CGXXXCGXXXCG-, MB-ODN 5/5, FIG. 2B ) is shown in FIG. 2. 395 oligonucleotides of -CGXXCGXXXCG- form and 354 oligonucleotides of -CGXXXCGXXXCG- form are present in chromosomal DNA of M. bovis BCG. In FIG. 1 , 20 nucleotide sequences were arranged in order of priority by giving a higher score as the frequency of XXCGXX was higher. Twenty nucleotide sequences were excluded from nucleotide sequences having CG at the 5'- or 3'-terminus, and 71 oligonucleotides for controlling candidate immune responses were selected and synthesized.

<Example 2>

Screening of MB-ODN with Immune Activity

<2-1> Immune Response of Synthesized Candidate MB-ODNs

MB-ODN prepared in Example <1-2> and various substitution variants thereof were examined to activate the promoters of IL-8 and IL-12 of macrophages.

a) culture of mouse macrophages

Raw 264.7 cells (ATCC, Manassas, VA) were cultured in DMEM medium with 10% FBS (Gibco BRL). Cell culture was performed in 37 ° C., 5% CO ₂ incubator (Forma).

b) IL-8 and IL-12 promoters Luc  Construction of Reporter Plasmids

To amplify the IL-8 promoter region (-135 bp to +46 bp), human genomic DNA was used as a template and PCR was performed using the following primer sets.

The amplified IL-8 promoter region fragment was inserted into pGL3-Basic plasmid (Promega) digested with Bgl II and Hin dIII. This produced the IL-8 promoter- Lu reporter plasmid (Wu GD et al., J. Biol. Chem. , 272: 2396-2403, 1997).

On the other hand, in order to amplify the IL-12 promoter region (-373 bp to +52 bp), human genomic DNA was used as a template and PCR was performed using the following primer set.

The amplified IL-12 promoter region fragment was inserted into pGL3-Basic plasmid (Promega) digested with Sac I and Xho I. This produced the IL-12 promoter- Lu reporter plasmid (Wu GD et al., J. Biol. Chem ., 272: 2396-2403, 1997).

c) Promoter Activation Assay: Luciferase Activity Assay

RAW 264.7 cells (ATCC, Rockviller, MID) were aliquoted into 12-well plates at a concentration of ⁵ × 10 ⁴ cells / well and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. The cells were co-transfected with the IL-8 promoter- Lu reporter plasmid or IL-12 promoter- Lu reporter plasmid prepared in b) and the pRL-null plasmid (Promega). Then, incubated for 24 hours at 37 ℃, 5% CO2 incubator. MB-ODNs (10 μg / well) described in FIG. 3 were treated in each well and incubated for 6 hours or 12 hours in a 37 ° C., 5% CO ₂ incubator. At this time, the control group was treated with PBS. Subsequently, PLB (passive lysis buffer) of the Dual-luciferase reporter assay system (Promega) was added to each well at a concentration of 100 μl / well to disrupt the cells. Luciferase assay was performed using the supernatant (15 μl) obtained by centrifuging the cell lysate. Luciferase activity was measured using a TD-20 / 20 (Turner designs) luminometer. The activity of each promoter following the treatment of MB-ODN is shown as relative to the control. That is, the activity of the control group is expressed as a fold activation.

As a result, as shown in Figure 4, the nucleotide sequence of MB-ODN4 / 5 # 31 was confirmed to activate the IL-8 promoter.

<2-2> Analysis of Activation of the IL-8 Promoter of MB-ODN4 / 5 # 31-like Sequences

The base sequence of MB-ODN4 / 5 # 31, which is effective for IL-8 promoter activation in Example <2-1>, has the same CGTTCGTGTCG sequence and the other nucleotide sequences similar to those of MB-ODN4 / 5 # 31 . Twenty nucleotide sequences present in bovis BCG chromosomal DNA were analyzed. As a result, as shown in Figure 5a it can be seen that there are 17 MB-ODN4 / 5 # 31 pseudo-base sequence. Thereafter, IL-8 promoter activity was measured by the same method as in Example <2-1>.

As a result, as shown in Figure 5b , it can be seen that the activity of activating the IL-8 promoter according to the base sequence. In addition to MB-ODN4 / 5 # 31 according to the present invention, the MB-ODN4 / 5 # 31.14 nucleotide sequence also showed high activity.

<Example 3>

Sequence Modification and Immune Responses of MB-ODN4 / 5 # 31 Oligonucleotides

<3-1> Sequence Modification of MB-ODN4 / 5 # 31 Oligonucleotide

The base sequence of MB-ODN4 / 5 # 31 was synthesized by modifying it as shown in FIG. 6A . The CG sequences of MB-ODN4 / 5 # 31 were replaced with GC sequences, respectively (# 31-CG-1, # 31-CG-2, # 31-CG-3). We also replaced the first and second CGs with GC bases (# 31-CG-4), the second and third CGs with GC bases (# 31-CG-5), and the first and third CGs. The first CG was changed to GC (# 31-CG-5). G of the CG sequence was changed to A, T, and C as shown in FIG. 6A , respectively. We also changed the first and second CGs to CAs, the second and third CGs to CAs, and the first and third and CGs to CAs.

<3-2> Investigation of Immune Responses of Sequence Modified MB-ODN4 / 5 # 31 Oligonucleotides

RAW 264.7 cells were placed in 12 well plates at 5 × 10 ⁴ cells / well and incubated in a 37 ° C. 5% CO ₂ incubator for 24 hours. After co-tranfection of the IL-8 promoter reporter plasmid and pRL-null plasmid, the cells were incubated in a 37 ° C. 5% CO ₂ incubator for 24 hours. Synthetic oligonucleotides were treated with 10 ug / well each and incubated in 37 ° C. 5% CO ₂ incubator for 6 hours. Thereafter, IL-8 promoter activity was measured by the same method as Example <2-1>.

As a result of measuring the activation by luciferase assay, synthetic oligonucleotides with any modified nucleotide sequences activated the IL-8 promoter of macrophages, 5'-AGCAGCGTTCGTGTGCGCCT-3 ', 5'-AGCAGCGTTCATGTCGGCCT-3' Oligonucleotides with the nucleotide sequence of 5′-AGCAGCGTTCGTGTCCGCCT-3 ′ highly activated the IL-8 promoter (FIG. 6B ). Other synthetic oligonucleotides showed lower IL-8 promoter activation than the control. The activation of IL-8 promoter was also measured by TTCATG, a variant of the second TTCGTG but not the CpG motif in oligonucleotides that activated IL-8 promoter. When the third CpG motif, GTCGGC, was modified, IL-8 promoter activation was increased by GTGCGC and GTCCGC sequences, again showing CpG motif ( FIG. 6 ).

<Example 4>

Investigation of immune response according to skeletal modification of MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 oligonucleotides

<4-1> Activation of RAW 264.7 Cells According to Skeletal Modification of MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14

RAW 264.7 cells were co-transfected with the IL-8- Lu promoter reporter vector or IL-12- Lu promoter reporter vector and pRL-null plasmid (Promega) produced in Example b). The transfected cells were treated with type O (phosphodiester backbone) and S type (phosphothioate backbone) MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 (0 or 10 μg / ml), respectively. Incubated for 8 hours. Thereafter, the IL-8 promoter and IL-12 promoter activity was measured in the same manner as in Example <2-1>. As a result, as shown in Figure 7, MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 according to the present invention showed the highest activity regardless of the shape of the skeleton (both type O and S) .

<4-2> NF- according to skeletal modification of MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 _K B activation

Cover glass was placed in a 24-well plate and RAM 264.7 cells were added ⁵ × 10 ⁵ cells / ml and incubated at 37 ° C. in a 5% CO ₂ incubator for 24 hours.

MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 were each treated with 5 ug / well. After 30 minutes, the cells were fixed with 3.7% formaldehyde and permeabilized with PBS containing 0.2% Triton-X 100. After blocking for 30 minutes with a solution containing 1% donkey serum in PBS (PBST) containing 0.2% Tween-20, 0.5 ul / well of mouse anti-p65 (titer 1: 500) antibody was added to PBST at room temperature for 2 hours. It was left to stand. After washing with PBST, it was treated with Donkey-anti-mouse-IgG-FITC (titer 1: 250) antibody for 2 hours. Confocal microscopy was used to observe migration to NF-kB nuclei (Lee, Y., et. Al., (2002) Blood 99, 4307-4317).

8A is a diagram illustrating the phenomenon of NF- _K B migration to the nucleus using confocal microscopy by staining NF- _K B by immunostaining. NF- _K B was located in the cytoplasm in the no-treated or non-CpG motif control (non-CpG-ODN 2041). When macrophages were treated with MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14, NF- _K B migrated to the nucleus. In MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 according to the present invention, NF-kB moved to the nucleus regardless of the shape of the skeleton (both O type and S type).

FIG. 8B is also an experiment confirming NF- _K B activation in RAW 264.7 cell lines treated with MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 by an Electrophoretic mobility shift assay (EMSA). RAW 264.7 cells were placed in 6 well plates at ⁵ × 10 ⁵ cells / ml and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 were each treated with 5 ug / well. After 30 minutes, the cells were reacted with nuclear extraction buffer and centrifuged to obtain nuclear protein. For EMSA, ³² P was labeled on a probe (5'-AGTTGAGGGGACTTTCCCAGGC-3 ') (SEQ ID NO: 13) having an NF- _K B binding position. A probe labeled ³² P and 20 ug of nucleoprotein were buffered (10 mM HEPES, pH 7.9, 65 mM NaCl, 1 mM dithiothreitol, 0.2 mM EDTA, 0.02% NP-40, 50 mg / ml poly (dIdC): poly (dIdC) and 8% glycerol) were mixed and reacted at room temperature for 30 minutes. The reaction solution was developed on 4% polyacrylamide gels containing 0.5X TBE (1X TBE is 89 mMTris borate and 1 mM EDTA, pH 8.0) and 2.5% glycerol. NF-kB competitor 5'-AGTTGAGGGGACTTTCCCAGGC-3 '(SEQ ID NO: 13) (Santa Cruz Biotechnology, Inc. , Santa Cruz, CA) was used, 50 times higher pretreatment was carried out EMSA. NF-kB antibody supershifi assays were performed with EMSA after reacting with lug NF- _K B antibody for 30 minutes at 4oC. 8 shows that EMSA activated NF- _K B in RAW 264.7 cells by MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14. MB-ODN4 / 5 # 31 and MB-ODN4 / 5 # 31.14 according to the present invention was confirmed by EMSA that NF- _K B is activated regardless of the shape of the skeleton (both type O and S).

<Example 5>

Induction of hematological immune response of MB-ODN4 / 5 # 31

<5-1> Immunization

Four-week-old Balb / c mice were intraperitoneally administered a mixture of hen egg lysozyme (HEL, 50 mg / mari) and MB-ODN4 / 5 # 31 (100 ug / mari). One week later, a second dose of the same amount of HEL and MB-ODN4 / 5 # 31 mixture was administered. After one week, blood was collected by heart punching, centrifuged to precipitate blood cells, and serum was obtained. ELISA was performed to confirm the titer of anti-HEL antibodies (total IgG, Ig G1, Ig G2a) from the obtained serum.

<5-2> ELISA

The obtained serum was diluted to 1:10 using PBS / 0.2% sodium azide and stored at -20 ° C. HEL (10 ug / ml sodium bicarbonate buffer pH 9.6) was added to 96 well immunoprate (Nunc) and left at 4 ° C. for 16 hours to attach HEL to the bottom of the plate. The plate was washed with PBST (PBS / 0.05% Tween 20) and 1% bovine serum albumin (BSA) was added to block for 1 hour at room temperature. Serum was serially diluted to 1: 3 using PBS, placed in the plate in order, and left at 4 ° C. for 16 hours and washed with PBST. Alkaline phosphatase-conjugated detecting antibody was mixed in PBST and placed in a plate and left at room temperature for 2 hours. 1: 2,000 goat anti-mouse Ig (H + L) (Southern Biotechnology Associates) antibody was used for total Ig detection. 1-StepTM ABTS (PIERCE) was added for color development and absorbance was measured at 405 nm using an ELISA reader (Labsystems) (Chu, RS, et.al., (1997) J. Exp. Med. 186, 1623 -1631).

Hematological immune responses were examined by injecting MB-ODN4 / 5 # 31 with hen egg lysozyme (HEL) intraperitoneally. MB-ODN4 / 5 # 31 fragment was found to have an adjuvant effect in hematologic immunity because the amount of antibody against HEL injected with MB-ODN4 / 5 # 31 is increased compared to HEL alone injection. 9). Freund's adjuvant, a drug in which extracts of Mycobacteria are mixed with paraffin oil, is one of the representative immunoadjuvant agents used for 60 years. However, the problem with this adjuvant is that cellular immunity is not achieved and cannot be used in humans. MB-ODN4 / 5 # 31 has been shown to be able to be used as a new adjuvant because it not only serves as an adjuvant to increase blood immunity but also stimulates immune cells to induce cellular immunity. MB-ODN4 / 5 # 31 has also been shown to be effective in the production of antibodies specific for Th1 immune response-specific IgG2a.

<Example 6>

Induced Cytokine Production by MB-ODN4 / 5 # 31

<6-1> Cytokine Expression Analysis in Dendritic Cells

a) Isolation of Dendritic Cells and MB-ODN4 / 5 # 31 Treatment

Progenitor cells were isolated from the bone marrow of the thighs of 4 week old Balb / c mice. RBC lysis solution (150 mM NH 4 Cl, 10 mM potassium carbonate, 0.1 mM EDTA pH 7.4) was reacted with the isolated progenitor cells, and the cells were recovered. The cells were dispensed in 6-well plates (Nunc) at a concentration of 2 × 10 ⁶ cells / well. In order to differentiate the progenitor cells of the bone marrow into dendritic cells, 10% FBS-containing RPMI medium containing 10 ng / ml of IL-4 and GM-CSF (biosource), respectively, was added to each well (Ghosh, M., J Immunol. 170: 5625-5629, 2003). The cells were cultured in an incubator containing 5% CO ₂ at 37 ° C. Cells were incubated for 6 days with changing medium every 2 days. Thereafter, 10 μg / ml of O-type MB-ODN4 / 5 # 31, CpG-ODN 1826, and non-CpG-ODN 2041 according to the present invention were treated.

b) Analysis of IL-12 Expression in Dendritic Cells

RT-PCR was performed to investigate the expression level of IL-12 in dendritic cells treated with O-type MB-ODN4 / 5 # 31 according to the present invention.

First, in Example <6-1a>, dendritic cells isolated from Balb / c mice were treated with O-type MB-ODN4 / 5 # 31 by time (0, 0.5, 1, 2, 4 and 8 hours). . The control group was treated with O type 1826 CpG ODN and 2041 non-CpG ODN, respectively.

Thereafter, total RNA was isolated from dendritic cells using TRIzol (Invitrogen). The total RNA (5 μg) was then treated with M-MLV reverse transcriptase (invitrogen) to prepare cDNA. Thus prepared cDNA as a template and PCR was performed using a primer set specific to IL-12 as follows.

PCR amplification was repeated 25 times with a cycle of DNA denaturation at 95 ° C for 30 seconds, primer annealing at 57 ° C for 40 seconds, and extension for 1 minute at 72 ° C. After the PCR reaction was completed, the PCR product amplified on a 1% agarose gel was confirmed. As a result, as shown in Figure 10, it was confirmed that the expression of IL-12 is induced only by O-type MB-ODN4 / 5 # 31 according to the present invention. On the other hand, unlike S-type 1826 CpG ODN reported to induce high levels of IL-12 expression (Lee, KW. Et al., Mol. Immunol. 41: 955-964, 2004), type 1826 The expression of IL-12 was not induced by CpG ODN.

<6-2> Analysis of IL-12 Expression in Mice by MB-ODN4 / 5 # 31

In order to investigate the expression level of IL-12p40 in mice treated with MB-ODN4 / 5 # 31 according to the present invention, ELISA was performed after immunization.

a) Immunization

Four-week old Balb / c mice were intraperitoneally administered MB-ODN4 / 5 # 31 and non-CpG-ODN 2041 (100 ug / mol) of type O and S, respectively. After 24 hours, blood was collected by heart punching and centrifuged to precipitate blood cells and serum was obtained.

b) ELISA

First, ELISA was performed to confirm the titers of anti-IL-12p40 and anti-IL-4 antibodies in serum isolated from Balb / c mice immunized with MB-ODN4 / 5 # 31 as in Example <5-2>. Was performed.

MB-ODN4 / 5 # 31 was injected intraperitoneally into mice to compare the production of IL-12p40 and IL-4. As a result, as shown in Figure 11a , MB-ODN4 / 5 # 31 of the present invention induced the production of IL-12p40, IL-4 was not changed. S-type MB-ODN4 / 5 # 31 increased IL-12 p40 production to higher levels. The present invention shows that MB-ODN4 / 5 # 31 has an effect of increasing Th1 immunoreactivity by increasing the production of IL-12p40.

<6-3> Analysis of IL-12 Expression in Mouse Spleen Immune Cells by MB-ODN4 / 5 # 31

Immune cells were collected from the spleens of mice and dispensed into each well at a concentration of 5 x 10 ⁵ cells / well. Thereafter, MB-ODN4 / 5 # 31, non-CpG-ODN 2041 (0 or 10 µg / ml) of type O or S was treated in each well and incubated for 24 hours. After the incubation was completed, the cell culture was separated. Then, in order to investigate the level of cytokines in the cell culture, sandwiches as in Example <5-2> using commercially available anti-IL-12 p40 and IL-4 antibodies (R & D systems, Minneapolis, Minn.) ELISA assays were performed respectively.

As a result, as shown in FIG. 11B, MB-ODN4 / 5 # 31 of the present invention increased IL-12 p40 production to high levels in splenic immune cells regardless of skeletal morphology. However, it did not affect the production of IL-4. In particular, IL-12, a representative cytokine that induces a Th1 immune response in the balance of Th1 / Th2 immune responses, was induced by the MB-ODN4 / 5 # 31 of the present invention, from which the MB-ODN4 / 5 # of the present invention. 31 was able to induce Th1 immune response.

<Example 7>

In vivo analysis to investigate the effects of atopic dermatitis treatment

<7-1> Application of MB-ODN4 / 5 # 31-containing ointment of the present invention

Six NC / Nga mice were divided into MB-ODN4 / 5 # 31 treated and untreated groups. Mice in the treatment group were applied ointment (0.2 mg / head) containing the prepared O-type MB-ODN4 / 5 # 31 to the atopic dermatitis lesion on the back of the mouse four times for two weeks once every five days. Untreated mice were coated with petrolatum to which the CpG ODN of the present invention was not added under the same conditions.

<7-2> Observation of lesion

On the 5th and 7th day after applying the MB-ODN4 / 5 # 31-containing ointment of the present invention, the lesion site of atopic dermatitis was visually observed. As a result, as shown in Fig. 12A , the loss of skin lesions was observed in the back of the mouse to which the O-type MB-ODN4 / 5 # 31 of the present invention was applied as compared with the untreated group of mice. In addition, the back skin of the mouse was removed, and the therapeutic effect of atopic dermatitis was examined by H & E staining. As a result, as shown in Figure 12b , hyperkeratosis and benign keratinosis (acanthosis) is significantly reduced in the lesions of mice coated with O-type MB-ODN4 / 5 # 31 of the present invention, the degree of infiltration of lymphocytes in the dermis It was confirmed that atopic dermatitis was treated by reduction.

<7-3> histological analysis

a) analysis of expression of cytokines

On the 5th, 7th and 14th day of application of the ointment containing the O-type MB-ODN4 / 5 # 31 of the present invention, skin of 1.51.5 cm 2 area was removed. Thereafter, the cells were fixed in a 4% formalin solution for at least one day. The fixed skin tissue was cut to a thickness of 5 μm by paraffin treatment. After the paraffin was removed, the experiment was performed according to the manual of LSAB + kit (DAKO, Denmark). 3% H ₂ O ₂ was treated for 10 minutes. Thereafter, blocking was performed by adding 10% normal goat serum diluted with TBS (Tris-buffered saline, pH7.4) containing 0.1% BSA. Subsequently, the first antibody-goat anti-mouse IL-10, goat anti-mouse IL-4 (Santa Cruz, USA) and rat anti-mouse IFN- (Pierce, USA)-were treated and reacted at 4 ° C. for at least 12 hours. I was. Thereafter, the biotin-labeled secondary antibody was reacted at room temperature for 30 minutes, and then streptavidin labeled with peroxidase was added and reacted at room temperature for about 30 minutes. After staining with DAB Substrate chromogen system (DAKO, Denmark) was observed under a microscope.

As a result, as shown in Figure 13 , the expression of IL-4 is reduced in the epidermis of the mouse 5 days after the application of the type O MB-ODN4 / 5 # 31 of the present invention, while the expression of IFN-gamma is increased It could be confirmed. This decreases the production of IL-4, a cytokine mediated by specifically high Th2 phenotype T lymphocytes in atopic dermatitis, whereas the type O MB-ODN4 / 5 # 31 of the present invention mediates cytology mediated by Th1 phenotype T lymphocytes. Increasing the production of the kine IFN-gamma indicates amelioration and treatment of symptoms of atopic dermatitis.

b) Cell number measurement of CD4 + and CD8 + lymphocytes

On the 5th, 7th and 14th day of application of the ointment containing the O-type MB-ODN4 / 5 # 31 of the present invention, skin of 1.51.5 cm 2 area was removed. The extracted skin tissue was frozen with liquid nitrogen. Then, it was inserted into a tissue-Tek OCT compound (Sakura Finetek USA, INC.) And cut to a thickness of 5 μm using a cryostat. The cleaved tissue was reacted with primary antibody-rat anti-mouse CD4 mAb (BD phamingen, USA) or rat anti-CD8 mAb (Serotec, UK) for 12 hours at 4 ° C. Thereafter, the biotin-labeled secondary antibody was reacted at room temperature for 30 minutes. Then, streptavidin labeled with peroxidase was added and reacted at room temperature for about 30 minutes. After staining with DAB Substrate chromogen system (DAKO, Denmark) was observed under a microscope. All pictures were taken 200 times.

As a result, as shown in FIG. 14 , it was confirmed that CD4 + and CD8 + lymphocyte cells were reduced in the skin of mice coated with O-type MB-ODN4 / 5 # 31 of the present invention. Reduction of CD4 + and CD8 + lymphocyte cells in lesions of atopic dermatitis is a very effective phenomenon in the treatment of atopic dermatitis (Christian V., et al. J Clin Invest. 104: 1097-1105, 1999).

<7-4> Serum IgE Level Analysis

Plasma was obtained from each group of mice and stored at −20 ° C. until use. Total IgE levels were measured using the mouse IgE BD OptEIA Kit (BD phamingen, USA). Then, in order to investigate plasma IgE antibody (BD pharmingen, USA) level, using a commercially available biotin-labeled IgE antibody (BD pharmingen, USA), sandwich ELISA analysis as in Example <5-2>, respectively Was performed.

As a result, as shown in FIG. 15 , serum IgE levels were significantly reduced in mice coated with the ointment containing the O-type MB-ODN4 / 5 # 31 of the present invention.

From these results, type O MB-ODN4 / 5 # 31 of the present invention increases the expression of cytokines mediated by Th1-lymphocytes, while inhibiting the expression of cytokines mediated by Th2-lymphocytes, thereby suppressing IgE in serum. Reducing the level was found to show a very good effect in the treatment of atopic dermatitis.

<Example 8>

Effect of Survival of Immune Cells on Irradiation MB-ODN4 / 5 # 31 Analysis of the effects of

<8-1> Expression of Bcl-xs / L by MB-ODN4 / 5 # 31 Treatment

RAW 264.7 cells were plated in 6 well plates at ₁ × 10 ⁵ cells / well and incubated in a 37 ° C. 5% CO ₂ incubator for 24 hours. Synthetic oligonucleotides were treated with 10 ug / well each and incubated in 37 ° C. 5% CO ₂ incubator for 6 hours. 100 ul / well of lysis buffer was added to the cells, and RAW 264.7 cells were crushed. Western blotting was performed using supernatant (15 ul) obtained by centrifugation of cell lysate. Treatment with antibody-goat anti-mouse Bcl-xs / L, reaction with a secondary antibody labeled with peroxidase, and Bcl- using an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA) xs / L was observed.

As a result, as shown in Figure 16 , MB-ODN4 / 5 # 31 according to the present invention acts on RAW264.7 cells to increase the Bcl-xs / L expression has the effect of increasing the cell survival rate have.

<8-2> Increased survival rate of macrophages by MB-ODN4 / 5 # 31 treatment

RAW 264.7 cells were incubated in a 4 well chamber slide (Lab-TEK Chamber slide, Nalge Nunc International, Inc) at 3 × 10 ⁴ cells / well and incubated in a 37 ° C. 5% CO ₂ incubator for 24 hours. Synthetic oligonucleotides were treated with 10 ug / well for 6 hours, 10 Gy irradiation with γ-irradiator, and incubated in 37 ° C. 5% CO ₂ incubator for 48 hours. 5x3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) solution (2ug / mL) was added directly to the medium to 0.4ug / mL to the cultured RAW 264.7 cells at 37 ° C. The reaction was carried out for 4 hours in a 5% CO ₂ incubator. After removing the medium of each well, 0.5 ml of DMSO was added, and the resulting formazan crystals were dissolved by reacting for 10 minutes at 37 ° C. Only 100ul of the reaction solution was taken and the absorbance was measured at 570 nm.

As a result, as shown in Fig. 17 , the treatment of MB-ODN4 / 5 # 31 according to the present invention shows that RAW 264.7 cells are inhibited from being killed by irradiation. Form O showed higher activity in the form of the backbone.

<8-2> Increase of survival rate of B cells by MB-ODN4 / 5 # 31 treatment

1 × 10 ⁵ cells / well of RPMI 8226 cells in 6-well plate and 10 ug / well of synthetic oligonucleotide for 6 hours, 10 Gy irradiation with γ-irradiator, followed by 37 ° C. 5% CO ₂ incubator for 48 hours. Incubated at. Propidium lodide (PI) was added to the cultured cells at a concentration of 50ug / mL and reacted on ice for 10 minutes, and then the degree of PI staining was confirmed by flow cytometry.

In addition, the cultured cells were washed twice with cold PBS, 5 μl of Annexin V-PE was added, and then reacted at room temperature for 15 minutes. After adding 0.4mL of Annexin V binding buffer, the degree of Annexin V binding was confirmed by flow cytometry.

As a result, as shown in Figures 18 and 19 , it shows that there is an effect of inhibiting the death of RPMI 8226 cells by irradiation by the treatment of MB-ODN4 / 5 # 31 according to the present invention.

From the above results, it can be seen that MB-ODN4 / 5 # 31 of the present invention has a very excellent effect in normalizing immune function by increasing normal immune cell survival rate in the treatment of refractory diseases such as cancer by irradiation.

Oligonucleotide fragments derived from Mycobacterium bovis BCG through the present invention having the configuration as described above play a role as an adjuvant in the formation of HEL antibodies and are involved in hematological immunity, and IL-8 and IL of macrophages -12 Promoter Activation In the present invention, the IL-8 promoter was found to be involved in the activation of innate immune cells. In addition, the oligonucleotide of the present invention not only acts as an adjuvant to increase hematologic immunity, but also stimulates immune cells to induce cellular immunity, and thus can be used as a new adjuvant. While ODN increases expression of cytokines mediated by Th1-lymphocytes in NC / Nga mice, an atopic dermatitis animal model, it inhibits the expression of cytokines mediated by Th2-lymphocytes, thereby reducing serum IgE levels. Through the treatment of atopic dermatitis was found to have a very good effect.

MB-ODN4 / 5 # 31 of the present invention was found to exhibit a very good effect in normalizing immune function by increasing the normal immune cell survival rate in the treatment of refractory diseases such as cancer by irradiation.

Claims (7)

delete An immune response enhancer (adjuvant) or an oligodeoxynucleotide for the treatment of immune diseases represented by the following general formula: [Formula]: DKMHKCGTTCRTGTCSGMYK (SEQ ID NO: 2) Wherein R is A or G and S is C or G, H is A, T or C, K is G or T, D is A, G or T, M is C or A, and Y is C or T. The oligodeoxynucleotide of claim 2, wherein the oligodeoxynucleotide survives normal immune cells upon radiation treatment. According to claim 2, wherein the oligodeoxynucleotide is oligodeoxynucleotide, characterized in that for treating or preventing skin diseases. The oligodeoxynucleotide of claim 2, wherein the oligodeoxynucleotide maintains a balance of various Th1 / Th2 immune responses. The oligodeoxynucleotide of claim 2, wherein the oligodeoxynucleotide is a phosphodiester bond or a phosphorothioate bond. According to claim 2, wherein the oligonucleotide is 5'-AGCAGCGTTCGTGTCGGCCT-3 '(SEQ ID NO: 3), 5'-AGCAGCGTTCATGTCGGCCT-3' (SEQ ID NO: 5), 5'-AGCAGCGTTCGTGTCCGCCT-3 '(SEQ ID NO: 6) Oligonucleotides, characterized in that selected from the group consisting of.

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