WO2006080596A1

WO2006080596A1 - Oligonucleotides derived from mycobacterium for stimulating immune function, treating immune-related diseases, atopic dermatitis and/or protecting normal immune cell

Info

Publication number: WO2006080596A1
Application number: PCT/KR2005/000266
Authority: WO
Inventors: Hyung-Joo Kwon; Tae-Yoon Kim; Doo-Sik Kim
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-01-28
Filing date: 2005-01-28
Publication date: 2006-08-03
Anticipated expiration: 2007-07-28
Also published as: CA2549423C; KR100980898B1; EP1841777A1; US20080249291A1; JP4836145B2; CA2549423A1; US7902161B2; KR20090096560A; CN100485034C; EP1841777A4; CN1926145A; EP1841777B1; JP2008501787A

Abstract

Disclosed are oligonucleotides for manipulating an immune reaction. The oligonucleotides of the present invention may be useful to stimulate the immune function, to treat the immune-related diseases and the atopic dermatitis, or to protect the normal immune cells.

Description

OLIGONUCLEOTIDES DERIVED FROM MYCOBACTERIUM FOR

STIMULATING IMMUNE FUNCTION, TREATING IMMUNE-RELATED

DISEASES, ATOPIC DERMATITIS AND/OR PROTECTING NORMAL

IMMUNE CELL

TECHNICAL FIELD

The present invention relates to oligonucleotides derived from Mycobacterium for manipulating immune reactions, which may stimulate immune reactions and

maintain a balance of the immune reactions and have an effect on various

immune-related diseases such as an atopic dermatitis, etc. in terms of a therapeutic use, and more specifically to oligonucleotides having three CpG motifs involved in stimulating immune reactions, which have an efficacy that is varied according to

modification of the DNA sequence(s), and may be used in treatment of various

immune-related diseases and an atopic dermatitis by stimulating immune reactions (an adjuvant) by the oligodeoxynucleotides in the form of phosphodiester and maintaining a

balance of the Thl/Th2 immune reactions, and also have an effect of increasing viability

of the cells as a treatment of the radiation.

BACKGROUND ART An immune system, generally initiated through an innate immune system, should be elaborately controlled to keep its balance. That is, the balances between immunity

and tolerance, between T helper type 1 (ThI) and T helper type 2 (Th2) immunities, and

between inflammation and unresponsiveness should be necessarily controlled elaborately. Unfortunately, conditions such as autoimmune-related diseases, allergic

diseases, chronic inflammation, etc. have, however, been spread since many therapeutic agents for the immune-related diseases developed up to now could not control the

immune system adequately. However, the innate immune system is a mechanism in

which immune cells are activated by recognizing a structural difference of a foreign substance (Pathogen-Associated Molecular Pattern, PAMP) when a pathogen was invaded, and subsequent signals are transmitted to initiate a cascade reaction of the

immune system, resulting in destruction of the pathogen. Accordingly, therapeutic

agents of the immune-related diseases should be necessarily developed to minimize the Evil mechanism after exact understanding of a Good & Evil mechanism using the innate

immune system.

In the 1890's, William B. Coley observed a surprising result that infection of

pathogenic microorganisms may induce an anti-cancer effect in cancer patients, and

therefore it was found that its modified bacterial therapy has about 40 % of the therapeutic effect if it is subject to 900 cancer patients. In the 1980's, Japanese

researchers recognized utility of Coley's toxin in a different and new aspect, proved that an active fraction of Bacillus Calmette-Guerin (BCG) shows an anti-cancer effect, and

confirmed that the anti-cancer activity of BCG is derived from an inherent

characteristics of DNA sequence. In 1995, Kreig, et al. proved, during the study of antisense oligonucleotides suppressing genes of a B cell, the fact that a synthetic

oligodeoxynucleotides (ODNs) of a specific DNA sequence composed of unmethylated

cytosine and guanine may induce activation of the immune cells. From the Kreig' s

aspect, it was newly presented that the anti-cancer effect of BCG proved by the Japanese researchers in the art is derived from the characteristics of unmethylated BCG DNA, and

the immunological activation by such a bacterial DNA allows the immune system of the vertebrate to distinguish self DNA and non-self DNA.

The early studies of the immunological activation and its control by bacteria focused on protein antigens such as Coley's toxin, which induces generation of the antibody. However, many of the studies reported that more powerful inducers for the

immunological activation are present among the components of the microorganism.

And. it was also proved that the bacterial DNA is prone to induce a powerful

immunological activation, and certain immune responses to each antigen (6, 7). An

CpG dinucleotide composed of two nucleic acid sequences is a gist of the

immunological activation and its control, and it was revealed from the recent studies that the vertebrate also distinguishes self DNA from bacterial DNA to activate the immune cells. Such a CpG motif is plentiful in the bacteria, but not in the vertebrate.

It was seen that an oligodeoxynucleotide including the CpG motifs

(CpG-oligodeoxynucleotide, CpG-ODN) activates various defense mechanisms of the host including innate immune responses and acquired immune responses (Akdis, CA.

Curr Opin Immunol., 12:641-646, 2000).

Recently, there has been developed a CpG-ODN whose backbone was modified

so as to increase usability of the CpG-ODN. The CpG-ODN with a phosphodiester backbone, referred to as a basic backbone of DNA, was easily decomposed in the body since it was sensitive to nucleases. Accordingly, the CpG-ODN has a low risk of

inducing in vivo toxicity. However, it is revealed that the CpG-ODN with the

phosphodiester backbone has a lower activity than the CpG-ODNs of the other backbones (Kwon, HJ. et al, Biochem. Biophys. Res. Commun., 311 :129-138, 2003).

On the other hand, the CpG-ODN with the phosphorothioate backbone was artificially

engineered by modifying its structure so that it cannot be decomposed in vivo by the

nuclease. The CpG-ODN with the phosphorothioate backbone has a good in vivo stability and an excellent ability to activate the B cells, compared to the CpG-ODN with

the phosphodiester backbone. Accordingly, the CpG-ODN modified into the

phosphorothioate backbone has been widely used lately. However, such a CpG-ODN

with the phosphorothioate backbone induces toxicity since it increase binding by the

ODN non-specific to many proteins, and therefore it is not easily decomposed in vivo. Also, it was reported that the CpG-ODN with the phosphorothioate backbone induces

the arthritis and exacerbates its symptoms (Deng GM et al, Arthritis & Rheumatism, 43

(2): 356-364, 2000), and causes the autoimmune-related diseases such as SLE (systemic

lupus erythematous) (Tanaka, T. et al, J Exp. Med. 175:597-607, 1992).

Formulations has been manufactured by adding various materials as the adjuvant to vaccine, and such a formulation has been designed to maximize an effect of the vaccine since the event of this century. However, aluminum salt (alum, Al₂O₃) is now

only an adjuvant approved so that it can be administered in the vaccine. In the recent

study, it was found that efficacy of the vaccine was much more excellent when a recombinant hepatitis surface antigen was mixed with the alum and the CpG-ODN and

administered to a mouse than when only the alum was used as the adjuvant (Davis H L.

et al, J. Immunol. 160: 870-876, 1998). It was seen that the alum slightly induces

cell-mediated immunity by inducing the Th2 immune reaction, while the CpG ODN

strongly induces humoral and cell-mediated immunity by inducing expression of the ThI cytokines. However, the problem is that the CpG-ODN used in this case may

cause a side effect since it has a phosphorothioate backbone.

Meanwhile, skin diseases are referred to as all abnormalities that appear in the skin of the animals including human. Amongst them, an atopic dermatitis has

characteristic major symptoms such as chronic/inflammatory skin diseases selected from

the group consisting of a serious pruritus, dry skins and an eczematous dermatitis

(Rudikoff, D. el aU Lancet. 351 : 1715-1721, 1998). Generally, the atopic dermatitis

tends to be inherited, and accompanied by an allergic asthma, an allergic rhinitis, an

allergic conjunctivitis and an urticaria, depending on individuals. A series of immunological abnormalities reported in the atopic dermatitis patients include an increased production of IgE, the reduced number and deteriorated function of CD8+

suppressor/cytotoxic T lymphocytes, the reduced number of ThI (T-cell Helper type 1) lymphocyte that secretes IFN-gamma, etc. Also, T lymphocyte having histological

CD4+ phenotype, infiltration of monocytes/macrophages, mast cells and eosinophils are

increased in the skin abnormality of the atopic dermatitis, and dendritic cells (DCs) and

epidermal Langerhans cells are also increased in the skin abnormality of the atopic dermatitis (Imokawa, G., et al., J. Invest. Dermatol, 96:523-526, 1991).

Many researchers have developed the methods for treating the cancer by killing

the cancer cells using X-ray. However, when the cancer is treated using the irradiation, cancer tissues and its adjacent immune cells all are inevitably damaged due to the

irradiation, resulting in its reduced immune functions. It has been reported that the

immune cells such as B cells (Ashwell JD et al, J. Immunol. 136:3649-3656, 1986), T cells (Prosser JS Int. J. Radial Biol. Relat. Stud. Phys. Chem. Med. 30:459-465, 1976), macrophages (Yoshihisa K et al, J. Radiat Res. 45:205-211, 2004), etc. were killed by

the irradiation (apoptosis). Accordingly, among the radiotherapeutic methods for treating the diseases such as a cancer, etc., there are required the methods that normal

immune cells except the cancerous cells are survived to normally maintain the immune reactions.

The present invention relates to oligonucleotides derived from Mycobacterium

bovis BCG for manipulating immune reactions, which may be used in treatment of various immune -related diseases by stimulating immune reactions (an adjuvant) and

maintaining a balance of the immune reactions, and also have effects of treating an

atopic dermatitis and increasing viability of the cells as a function of the irradiation.

DISCLOSURE OF INVENTION

Accordingly, the present invention provides CpG oligodeoxynucleotides isolated

from the Mycobacterium bovis BCG (MB-ODN), which are presented in following General Formula and composed of DNA sequences including at least two unmethylated

CpG motifs, wherein the CpG oligodeoxynucleotides may be used to stimulate immune reactions (an adjuvant), maintain a balance of Thl/Th2 immune reactions so as to treat

various immune-related diseases, and protect normal immune cells when intractable diseases such as a cancer, etc. are treated using the radiotherapy, and also provides a method for treating or preventing skin diseases.

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

wherein, R represents A or G; S represents C or G; H represents A, T or C; K

represents G or T; and M represents C or A. In the present invention, the oligonucleotides preferably further include five

nucleotides, presented in following General Formula, at a 5 '-terminal end and a

3 '-terminal end:

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

wherein, R represents A or G; S represents C or G; H represents A, T or C; K

represents G or T; D represents A, G or T; M represents C or A; M represents C or A; and Y represents C or T.

In the present invention, the term 'CpG motif means a DNA sequence that includes unmethylated cytosine-guanine dinucleotides (referred to as unmethylated

cytosine-phosphate-guanine dinucleotides) connected by phosphodiester bond

(phosphate bond), and activates immune reactions. Also, the term 'CpG

oligodeoxynucleotide (hereinafter, referred to as 'CpG-ODN')' means an oligodeoxynucleotide that includes at least two CpG motifs.

Also in the present invention, the term "subject' means a mammal, particularly an animal including human. The subject may be a patient in need of treatment.

In the present invention, the oligonucleotides is preferably selected from the

group consisting of 5'-AGCAGCGTTCGTGTCGGCCT-S' (SEQ ID NO: 3),

5 '-AGCAGCGTTCGTGTGCGCCT-S' (SEQ ID NO: 4),

5 '-AGCAGCGTTCATGTCGGCCT-S' (SEQ ID NO: 5),

5'-AGCAGCGTTCGTGTCCGCCT-S ' (SEQ ID NO: 6),

5'-GTATTCGTTCGTGTCGTCCT-S ' (SEQ ID NO: 7), and

5'-TGACTCGTTCGTGTCGCATG-S ' (SEQ ID NO: 8).

The MB-ODN of the present invention may be derived from natural sources (for example, chromosomal DNA of M. bovis BCG), and chemically synthesized, or recombinantly manufactured. The MB-ODN of the present invention may be

synthesized using various techniques and apparatuses for synthesizing the nucleic acid,

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 U.S Patent No. 4,458,066).

The MB-ODN of the present invention preferably has a phosphodiester backbone. The phosphodiester backbone, referred to as a basic backbone of DNA, has

a low risk of inducing in vivo toxicity since it is easily decomposed in vivo by the nucleases. The MB-ODN of the present invention is characterized in that it has an

excellent immunological activity in vitro and in vivo unlike the other conventional CpG ODNs although it has the phosphodiester backbone. Also, the MB-ODN of the present invention may include modified backbones. It was revealed that modification of the

oligonucleotide backbone might allow the CpG ODN to strengthen the activity and/or

stability when the CpG-ODN is administered in vivo. In the MB-ODN of the present invention, the preferred modification of the backbone includes modification into phosphorothioate, which is allowed to be resistant to its decomposition. The

modification into phosphorothioate may be generated at the terminal ends, and for

example two or three of the last 5' or 3' nucleotides may be connected by phosphorothioate bonds. Also, the MB-ODN of the present invention may be modified

to have a secondary structure (for example, a stem-loop structure) so that it can be

resistant to its decomposition. Preferably, the MB-ODN of the present invention may

be modified to have a partially phosphorothioate-modified backbone. The phosphorothioate may be synthesized by the automatic techniques using phosphoramidate or H-phosphonate chemistry (S. E. Beaucage et ah, Tetrahedron Lett.,

22:1859. 1981 ; Froehler et ah, Nucl Acid. Res., 14:5399-5407). As another modification example, aryl- and alkyl-phosphonate may be synthesized, for example as

described in U.S. Patent No. 4,469,863, and alkylphosphotriester (a charged oxygen

residue is alkylated, as described in U.S. Patent No. 5,023,243 and EP Patent No.

092,574) may be manufactured by an automatic solid-phase synthesis using commercially available reagents. Also, still another modification example, which makes the MB-ODN less sensitive to the decomposition, includes acetyl-, thio- and

similar modifications of adenosine, cytosine, guanine, thymine and uridine, as well as atypical bases such as inosine and quesine. The CpG-ODN having diols such as

tetraethylglycol or hexaethyleneglycol at the terminal ends is also more resistant to its

decomposition. In addition, the CpG-ODN further includes combination of

phosphodiester and phosphorothioate, phosphotriester, phosphoramidate, methylphosphonate, methylphosphorothionate, phosphorodithoate and combinations thereof (Khorana et al, J. Molec. Biol, 72:209, 1972; Goodchild, J. Bioconjugate

Chem.. 4: 165, 1990). As described above, the CpG-ODN having the modified backbone may have stronger immunological effects by means of enhanced nuclease

resistance, increased cellular uptake, increased protein uptake and/or altered intracellular localization, etc.

The preferred backbone of the MB-ODN of the present invention is a

phosphodiester (hereinafter, referred to as "O-type") or phosphorothioate (hereinafter,

referred to as "S-type") backbone, and the most preferred backbone is the O-type backbone that is not easily decomposed in vivo to induce side effects.

It was seen that the MB-ODN according to the present invention strongly

induces the humoral immune reactions by inducing expression of the ThI cytokines, and

has an adjuvant activity that improves efficiency of the vaccine. Specific physiological activities are as follows:

1) Production of IL- 12 is increased in immune cells from a mouse and a mouse

spleen.

2) Dendritic cells are activated to induce expression of the IL- 12.

3) Production of antibodies is increased when HEL and the MB-ODN are used as an antigen and an adjuvant, respectively. At this time, it is revealed that production

of IgG2a is more increased as a result of the ThI immune reaction when CFA is used as an antigen.

The MB-ODN according to the present invention has an effect of improving the efficiency of the vaccine by means of the activities as described above. Unlike the

conventional CpG-ODNs known in the prior art, the MB-ODN of the present invention is characterized in that it has nearly the same activity regardless of its backbone shapes.

In the present invention, it was revealed that the CpG-ODN of the present invention

modified into an O-type backbone has nearly the same activity as the CpG-ODN modified into an S-type backbone. Also, the CpG-ODN of the present invention may be effectively used as the adjuvant of the vaccine since it was revealed that it strongly induces the humoral immune reactions by inducing expression of the ThI cytokines.

The MB-ODN according to the present invention has the physiological activities

that control balance of the Thl/Th2 immune reaction by suppressing the Th2 cytokine (for example, IL-4), and/or inducing the ThI cytokine (for example, IL- 12). Specific physiological activities are as follows: 1) Macrophages are activated to activate an IL- 12

promoter; 2) Dendritic cells are activated to induce expression of the IL- 12; 3)

Production of the IL- 12 is increased in a mouse; 4) Production of the IL- 12 is increased

in immune cells of a mouse spleen; 5) Expression of cytokines (IL-4 and IL-10)

mediated by Th2-lymphocytes is inhibited; 6) The cell number of CD4+ and CD8+ lymphocytes is reduced in a lesion site of the atopic dermatitis; and 7) A level of IgE is reduced in blood serum.

The MB-ODN according to the present invention has effects of treating the skin

diseases or improving their symptoms by means of the activities as described above. Unlike the conventional CpG-ODNs known in the prior art, the CpG-ODN of the

present invention is characterized in that it has nearly the same activity regardless of its backbone shape. In the present invention, it was revealed that the CpG-ODN of the

present invention modified into an O-type backbone has nearly the same activity as, or

the more excellent activity than the CpG-ODN modified into an S-type backbone. Therefore, the MB-ODN of the present invention may be useful to treat or prevent all

the skin diseases. Also, the CpG-ODN of the present invention may be effectively used as a therapeutic agent of the immune-related diseases (for example, an asthma) that

appear due to unbalance of the Thl/Th2 immune reaction since the balance of theThl/Th2 immune reaction is maintained by inducing expression of the ThI cytokines.

The MB-ODN according to the present invention has an effect of increasing

viability of the immune cells. The MB-ODN has effects of stimulating macrophages to increase expression of Bcl-xs/L, and then inhibiting the apoptosis caused by the irradiation. Also, the MB-ODN has an effect of then inhibiting the apoptosis of the B

cells caused by the irradiation. Accordingly, the MB-ODN may be effectively used to

normalize the immune functions by increasing the viability of the normal immune cells when intractable diseases such as a cancer, etc. are treated using the irradiation.

Specific physiological activities of the MB-ODN are as follows: 1) Expression of Bcl-xs/L is increased in the macrophages; 2) Viability of the macrophages is increased

using the irradiation; and 3) Viability of the B cells is increased using the irradiation.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of preferred embodiments of

the present invention will be more fully described in the following detailed description,

taken accompanying drawings, incorporated herein in its entirety by this reference. In the drawings: Fig. 1 is a result of analyzing chromosomal DNA sequences of E. coli and M hovis BCG using a computer program. All DNA sequences (CpG motifs) in which

two bases are present in both terminal ends of a CG dinucleotide are analyzed. As a result, it was confirmed that much more numbers of the CpG motifs are present in the chromosomal DNA of M bovis BCG, as shown in Fig. 1. Fig. 2 is a result of analyzing DNA sequences in which three CpG motifs are present on 20 base pairs among the DNA sequences present in the chromosomal DNA

of M. bovis BCG. In the CpG motifs, the oligonucleotides have 4 and 5 base gaps between the bases C and C (-CGXXCGXXXCG-, MB-ODN 4/5), and have each of 5 base gaps between the bases C and C (-CGXXXCGXXXCG-, MB-ODN 5/5). It is

shown that 395 oligonucleotides in the form of -CGXXCGXXXCG- and 354

oligonucleotides in the form of -CGXXXCGXXXCG- are present in the chromosomal DNA of M bovis BCG.

Fig. 3 is a table showing that 71 candidate oligonucleotides for controlling the immune reactions are selected and synthesized, and the used for detecting the candidate sequences.

Fig. 4 is a diagram showing activation of IL-8 and IL- 12 promoters in RAW

264.7 cells treated with the 71 oligonucleotides for controlling the immune reactions,

synthesized in the form of the phosphodiester bond as shown in Fig. 3. Fig. 4a is a

diagram showing a result of comparing how much 35 synthesized oligonucleotides in

the MB-ODN 4/5 form activate an IL-8 promoter of the macrophage, Fig. 4b is a diagram showing a result of comparing how much 35 synthesized oligonucleotides in the MB-ODN 4/5 form activate an IL- 12 promoter of the macrophage, and Fig. 4c is a

the MB-ODN 5/5 form activate an IL-8 promoter of the macrophage.

Fig. 5 is a diagram showing a result of selecting 17 oligonucleotides having five different DNA sequences toward each of 5' end and 3' end of the core

CGTTCGTGTCG of MB-ODN 4/5#31 present on 20 base pairs among the DNA sequences present in the chromosomal DNA of M. bovis BCG (Fig. 5a), and then

synthesizing the oligonucleotides with the phosphodiester backbones to compare how

much the 17 oligonucleotides activate an IL-8 promoter of the macrophage (Fig. 5b).

Fig. 6 is a diagram showing a result of comparing how much the oligonucleotides MB-ODN 4/5#31 (M) in which the base number of the MB-ODN

4/5#31 is reduced to 15 base pairs, #31 -CG in which CG sequences are substituted with GC sequences, and #31 -A, B, C, D in which G of the CG sequences are independently substituted with A, T, or C (Fig. 6a) activate an IL-8 promoter of the macrophage (Fig. 6b).

Fig. 7 is a diagram showing that the backbones of the MB-ODN 4/5#31 and the

#31.14 are synthesized in the forms of phosphodiester and phosphorothioate to compare how much they affect activation of IL-8 and IL- 12 promoters in a mouse macrophage

cell line RAW 264.7. Figs. 7a and b are diagrams showing that the backbones of the

MB-ODN 4/5#31 are synthesized in the forms of phosphodiester and phosphorothioate

to activate the IL-8 promoter in a concentration-dependant manner. Figs. 7c and d are diagrams showing that the backbones of the MB-ODN 4/5#31 and the #31.14 are

synthesized in the forms of phosphodiester and phosphorothioate to compare how much

they affect activation of IL-8 and IL- 12 promoters.

Fig. 8 is a diagram showing that NF-κB is activated when the RAW 264.7 cell lines are stimulated with the phosphodiester and phosphorothioate backbones of the

MB-ODN 4/5#31. Fig. 8a is an confocal microscopic photograph showing that

localization of NF-κB is confirmed by treating and fixing the RAW 264.7 cells with MB-ODN 4/5#31 (10 μg/ml) for 30 minutes, followed by conducting an indirect

immunofluorescence assay using the NF-κB p65-specific antisera. Fig. 8b shows a

result of an electrophoretic mobility shift assay (EMSA) in which the RAW 264.7 cells

are treated with MB-ODN 4/5#31 (10 μg/ml) for 30 minutes, and then nucleoproteins

are isolated to confirm binding of the NF-κB to a NF-κB consensus binding site. Fig. 9 is a diagram showing that the backbones of the MB-ODN 4/5#31 are synthesized in the forms of phosphodiester and phosphorothioate to compare how much they affect the humoral immunity of the Balb/c mice abdominally immunized by hen

egg lysozyme (HEL).

Fig. 10 is an electrophoretic diagram showing that an effect of the MB-ODN

4/5#31 of the present invention is compared to those of the conventional 1826

CpG-ODN and non-CpG-ODN (2041) for expression of the IL- 12 in the dendritic cells.

Fig. 11 is a diagram showing that an effect of the modified backbones of the

MB-ODN 4/5#31 according to the present invention is compared to those of the

conventional 1826 CpG-ODN and non-CpG-ODN (2041) for production of the IL- 12 p40. Fig. l la is a diagram confirming how much the IL- 12 p40 is produced in the

blood serum after the Balb/c mice is abdominally immunized with the MB-ODN 4/5#31.

Fig. l l a is a diagram confirming a level of the produced IL- 12 p40 when spleen

immune cells is separated from the Balb/c mice, and then treated with the MB-ODN

4/5#31.

Fig. 12 is a photograph showing that the atopic dermatitis is treated by administration of the O-type MB-ODN 4/5#31 according to the present invention using

the animal model.

Fig. 12a is a photograph showing that a NC/Nga mouse is examined with the naked eye on 5 and 7 days after the O-type MB-ODN 4/5#31 of the present invention is applied to an atopic dermatitis lesion present in the back of the NC/Nga mouse; and Fig.

12b is a photograph showing that the O-type MB-ODN 4/5#31 is applied to the dorsal skin of the NC/Nga mouse in which the atopic dermatitis is broken out, extracted out and then stained with H&E stain. In the drawing, "<-»^■" indicates a lesion site of an

acanthosis, and '"— ►" indicates a lesion site of an hyperkeratosis.

Fig. 13 is a microscopic photograph showing a result of histochemical analysis

where a level of the expressed cytokines (IL-4 and IFN-gamma) is observed in the

dorsal skin of the NC/Nga mouse to which the O-type MB-ODN 4/5#31 of the present

invention is administered. In the drawing, the arrows indicate sites of the expressed cytokines.

Fig. 14 is a microscopic photograph showing a result of histochemical analysis where the cell number of the CD4+ and CD8+ lymphocytes is observed in the dorsal

skin of the NC/Nga mouse to which the O-type MB-ODN 4/5#31 of the present invention is administered.

Fig. 15 is a diagram showing a level of the IgE present in the blood serum of the

NC/Nga mouse to which the O-type MB-ODN 4/5#31 of the present invention is

administered. In the drawing, "AD-" represents an untreated group. Fig. 16 is a diagram showing using a Western blotting that expression of the

Bcl-xs/L is increased when the macrophage cell line RAW264.7 is treated with the

O-type MB-ODN 4/5#31.

Fig. 17 is a diagram showing, using an MTT assay, that viability of the RAW264.7 cells is increased when the RAW264.7 cells are irradiated with the radiation after the RA W264.7 cells are pre-treated with the MB-ODN 4/5#31.

Fig. 18 is a diagram showing, using an flow cytometry after PI staining, that

viability of the RPMI 8226 cells is increased when the B cell line RPMI 8226 is

irradiated with the radiation after the RPMI 8226 cells are pre-treated with the MB-ODN 4/5#31.

Fig. 19 is a diagram showing, using an flow cytometry after Annexin V staining,

that viability of the RPMI 8226 cells is increased when the B cell line RPMI 8226 is

irradiated with the radiation after the RPMI 8226 cells are pre-treated with the

MB-ODN 4/5#31.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereinafter, preferred embodiments of the present invention will be described in

detail with reference to the accompanying drawings.

Therefore, the description proposed herein is just a preferable example for the

purpose of illustrations only, not intended to limit the scope of the invention.

Analysis of DNA Sequences of Chromosomal DNAs from E. coli and M. bovis

BCG

<1-1> Analysis of DNA Sequences of CpG Motifs in the Chromosomal DNAs from E. coli and M. bovis BCG

The present inventors analyzed chromosomal DNA sequences of E. coli and M. bovis BCG using a computer program. The frequency of DNA sequences composed of 6 nucleotides, present in the chromosomal DNAs of E. coli and M. bovis BCG, was

calculated using the computer program. It was found that the probability of the DNA

sequence XXCGXX on the chromosomal DNA is theoretically 1/4⁶, but the probability

of the sequence XXCGXX in the chromosomal DNAs of E. coli and M. bovis BCG is actually much higher. Also, it was confirmed that frequency of the sequence

XXCGXX in the chromosomal DNA of M. bovis BCG is more higher than that of E. coli (Fig. 1 ).

<l -2> Analysis of DNA Sequence of CpG ODN in Chromosomal DNA from M. bovis BCG

20 base pairs of Oligonucleotides were randomly selected from M. bovis BCG

chromosomal DNA, and then the oligonucleotides including the three motifs XXCGXX were selected among them.

For example: GACGTTGAGTCGTTAACGAG

The results of analyzing the oligonucleotides having 4 and 5 base gaps between

C and C (-CGXXCGXXXCG-, MB-ODN 4/5, Fig. 2a), and the oligonucleotides having

each of 5 base gaps between C and C (-CGXXXCGXXXCG-, MB-ODN 5/5, Fig. 2b) is listed, as shown in Fig. 2. It was shown that 395 oligonucleotides in the form of

-CGXXCGXXXCG- and 354 oligonucleotides in the form of -CGXXXCGXXXCG- are

present in the chromosomal DNA of M. bovis BCG. 20 base pairs of the oligonucleotides were listed on the order of priority by giving high marks to the

oligonucleotides including the high frequencies of the motif XXCGXX, as shown in Fig. 1. The oligonucleotides whose CG is present in the 5'- or 3'- terminal end of 20 base pairs of the oligonucleotides was excluded, and then the 71 candidate oligonucleotides for controlling the immune reaction were selected, synthesized and used for detecting

the candidate substances.

<Example 2> Detection of MB-ODN Having Immune Activity

<2-l> Immune Reaction of Synthesized Candidate MB-ODNs

It was examined whether or not the MB-ODNs prepared in the Example <l-2>,

and their various substituents could activate the IL-8 and IL- 12 promoters of the

macrophages. a) Cultivation of Mouse Macrophage

Raw 264.7 cells (ATCC, Manassas, VA) were cultured in a DMEM medium

including 10 % FBS (Gibco BRL). Cell culture was carried out in a 5 % CO₂

incubator (Forma) at 37 ^°C .

b) Design of IL-8 and IL- 12 promoter-Luc Reporter Plasmid

In order to amplify an IL-8 promoter region (from -135 bp to +46 bp), human

genome DNA was used as a template, and following primer sets were used to conduct a

PCR reaction.

5' primer (SEQ ID NO: 9) 5'- GTGAGATCTGAAGTGTGATGACTCAGG-3' 3' primer (SEQ ID NO: 10) 5'- GTGAAGCTTGAAGCTTGTGTGCTCTGC-3'

A fragment of the amplified IL-8 promoter region was inserted into a

pGL3-Basic plasmid (Promega) digested by the restriction enzymes BgHl and Hinάlll.

Therefore, an IL-8 promoter-lMC reporter plasmid was constructed (Wu G. D. et al , J

Biol Chem., 272:2396-2403, 1997).

Meanwhile, in order to amplify an IL-12 promoter region (from -373 bp to +52

bp), human genome DNA was used as a template, and following primer sets were used

to conduct a PCR reaction. 5' primer (SEQ ID NO: 1 1 ) 5¹- CATGAGCTCAGCCTCCCGTCTGACC-3'

3' primer (SEQ ID NO: 12) 5'- CTGGGCTCGAGGGAGAGTCCAATGG-3'

A fragment of the amplified IL- 12 promoter region was inserted into a pGL3 -Basic plasmid (Promega) digested by the restriction enzymes Sac I and Xho I. Therefore, an IL- 12 promoter-Zwc reporter plasmid was constructed (Wu G. D. et al, J

Biol Chem., 272:2396-2403, 1997).

c) Analysis of Promoter Activation: Luciferase Activity Assay RAW 264.7 cells (ATCC, Rockviller, MID) were divided into 12-well plates at a

concentration of 5X10⁴ cells/well and cultured at 37 ^°C for 24 hours in a 5 % CO₂

incubator. The cells were co-transfected with the IL-8 promoter-Z,«c reporter plasmid or the IL- 12 promoter-Lwc reporter plasmid, which were constructed in the b), and a

pRL-null plasmid (Promega). Then, the co-transfected cells were cultured at 37 ^°C

for 24 hours in the 5 % CO₂ incubator. Each well was treated with the MB-ODNs (10

μg/well) shown in the Fig. 3, and cultured at 37 ^°C for 6 hours or 12 hours in the 5 %

CO₂ incubator. At this time, the control group was treated with PBS. Then, PLB

(passive lysis buffer) of a dual-luciferase reporter assay system (Promega) was added to

each well at a concentration of 100 μl/well to homogenize the cells. The cell lysates was centrifuged, and the resultant supernatant (15 μl) was used to conduct a luciferase assay. The luciferase activity was measured using a TD-20/20 (Turner designs) luminometer. Each promoter activity by treatment of the MB-ODNs was measured as

a relative activity of the control group. That is, if activity of the control group was set

to T, then activities of the experimental groups were presented as fold activation of the control group.

As a result, it was confirmed that the DNA sequence of the MB-ODN4/5#31 activates the IL-8 promoter, as shown in Fig. 4.

<2-2> Activation of IL-8 Promoter by Oligonucleotides Homologous to MB-ODN4/5#31

20 base pairs of oligonucleotides, present in the chromosomal DNA of M. bovis BCG and homologous to the MB-ODN4/5#31 , were analyzed, the homologous oligonucleotides having different DNA sequences except that they have the sequence

CGTTCGTGTCG within the DNA sequences of MB-ODN4/5#31 having the effect of

the IL-8 promoter activation, as shown in the Example <2-l>. As a result, it was seen that 17 oligonucleotides homologous to the MB-ODN4/5#31 are present, as shown in Fig. 5a. And then, the same method as in the Example <2-l> was repeated to measure the IL-8 promoter activity.

Accordingly, it was revealed that the ability to activate the IL-8 promoter is

varied depending on the DNA sequences, as shown in Fig. 5b. It was seen that the MB-ODN4/5#31.14 also has high activity, in addition to the MB-ODN4/5#31 according to the present invention.

Modification and Immune Reaction of the DNA Sequence of the

Oligonucleotide MB-ODN4/5#31

<3-l> Modification of DNA Sequence of the Oligonucleotide MB-ODN4/5#31 The DNA sequence of the oligonucleotide MB-ODN4/5#31 was modified to synthesize DNA sequences, as described Fig. 6. Each CG sequence of the MB-ODN4/5#31 was changed to a GC sequence (#31 -CG-I, #31-CG-2, #31-CG-3).

Also, the first CG and the second CG were changed to a GC sequence (#31-CG-4), the

second CG and the third CG were changed to a GC sequence (#31-CG-5), and the first

CG and the third CG were changed to a GC sequence (#31 -CG-5). Each base G of the CG sequences was changed to A, T and C, respectively, as shown in Fig. 6a. Also, the

first and second CG was changed to the sequence CA, and the second and third CG was

changed to the sequence CA, and the first and third CG was changed to the sequence CA.

<3-2> Measurement of Immune Reactions by Oligonucleotides Modified from

the Oligonucleotide MB-ODN4/5#31

5x10⁴ cells/well of RAW 264.7 cells were spread on a 12-well plate, and incubated at 37 ^°C for 24 hours in a 5 % CO₂ incubator. An IL-8 promoter reporter

plasmid and a pRL-null plasmid were co-transfected, and then incubated at 37 ^"C for 24

hours in a 5 % CO₂ incubator. Each well was treated with synthetic oligonucleotides at 10 ug/well, and incubated at 37 ^°C for 6 hours in a 5 % CO₂ incubator. Then, the same

method as in the Example <2-l> was repeated to measure the IL-8 promoter activities.

The luciferase assay was used for measuring how much the synthetic oligonucleotides having any modified DNA sequences activates the IL-8 promoter of the macrophage. As a result, the IL-8 promoter was highly activated by the

oligonucleotides 5 ' -AGC AGCGTTCGTGTGCGCCT-3 ' ,

5'-AGCAGCGTTCATGTCGGCCT-S ' 5'-AGCAGCGTTCGTGTCCGCCT^ ' (Fig. 6b). Other synthetic oligonucleotides showed lower IL-8 promoter activities than the control group. In the oligonucleotides that activate the IL-8 promoter, the IL-8

promoter activities were measured even by the oligonucleotide having the second CpG

motif TTCGTG variant "TTCATG", which is not the CpG motif. It was revealed that

when the third CpG motif "GTCGGC" was modified, the sequences GTGCGC and

GTCCGC reappearing in the CpG motif could activate the IL-8 promoter (Fig. 6).

Examination of Immune Reaction by Backbone Modifications of

Oligonucleotides MB-ODN4/5#31 and MB-ODN4/5#31.14

<4-l> Activation of RAW 264.7 Cells by Backbone Modifications of

MB-ODN4/5#31 and MB-ODN4/5#31.14

RAW 264.7 cells were co-transfected with ΪL-8-Luc promoter reporter vector or \L-\2-Luc promoter reporter vector constructed in the step b) of the Example <2-l>, and

pRL-null plasmid (Promega). The transfected cells were treated with the O-type

(phosphodiester backbone) and S-type (phosphorothioate backbone) MB-ODN4/5#31 and MB-ODN4/5#31.14 (0 or 10 μg/ml), and incubated for 8 hours, respectively. Then,

the same method as in the Example <2-l> was repeated to measure activities of the IL-8 promoter and the I L- 12 promoter. As a result, the oligonucleotides MB-ODN4/5#31 and MB-ODN4/5#31.14 according to the present invention showed the highest activities

regardless of the backbone shapes (both of O-type and S-type), as shown in Fig. 7.

<4-2> Activation of NF-κB by Backbone Modifications of MB-ODN4/5#31 and

MB-ODN4/5#31.14

A cover glass was put on a 24-well plate, and then RAM 264.7 cells was added at a concentration of 5 x 10⁵ cells/ml and incubated at 37 ^°C for 24 hours in a 5 % CO₂

incubator. Each well was treated with the oligonucleotides MB-ODN4/5#31 and MB-ODN4/5#31 .14 at a quantity of 5 ug/well. After 30 minutes, the cells were

immobilized using 3.7 % formaldehyde, and then permeabilized with PBS including 0.2 % Triton-X 100. The cells were blocked for 30 minutes in a solution in which 1 %

donkey serum was added to PBS (PBST) including 0.2 % Tween-20, and then 0.5 ul/well of mouse anti-p65 (titer 1 : 500) antibodies was added to PBST and kept at room

temperature for 2 hours. After the cells were washed with PBST, they were treated

with Donkey-anti-mouse-IgG-FITC (titer 1 :250) antibodies for 2 hours. Mobilization of the NF-κB into nuclei was observed using a confocal microscopy (Lee, Y., et. al.,

(2002) Blood 99, 4307-4317).

Fig. 8a is a photograph showing that NF-κB was stained using an immuno staining method and mobilization of the NF-κB into nuclei was observed using the confocal microscopy. The NF-κB was present in cytoplasm in an untreated control

group or a CpG motif-free control group (non-CpG-ODN 2041). When the macrophages were treated with the MB-ODN4/5#31 and the MB-ODN4/5#31.14, the NF-κB was mobilized into the nuclei. The oligonucleotides MB-ODN4/5#31 and

MB-ODN4/5#31.14 according to the present invention were mobilized into the nuclei regardless of the backbone shapes (both of O-type and S-type). Fig. 8b is an electrophoretic diagram showing that NF-κB is activated in RAW

264.7 cell lines treated with MB-ODN4/5#31 and MB-ODN4/5#31.14 using an

electrophoretic mobility shift assay (EMSA). 5 x 10⁵ cells/ml of RAW 264.7 cells were added to each 6-well plate, and incubated at 37 ^°C for 24 hours in a 5 % CO₂ incubator. Each cell was treated with the oligonucleotides MB-ODN4/5#31 and MB-ODN4/5#31.14 at a quantity of 5 ug/well. After 30 minutes, the cells were reacted

in a nuclear extraction buffer, and then centrifuged to obtain nucleoprotein, which was used for conducting the EMSA. The probe

(5'-AGTTGAGGGGACTTTCCCAGGC-S') (SEQ ID NO: 13), which has a NF-κB binding site, was labeled with ³²P for the EMSA. The ³²P-labeled probe and 20 ug of

the nucleoprotein were mixed in a buffer (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 (dldC) and

8 % glycerol), and then reacted at room temperature for 30 minutes. The reaction

solution was electrophoresed in a 4 % polyacrylamide gel including 0.5X TBE (IX TBE is 89 mMTris borate and 1 mM EDTA, pH 8.0) and 2.5 % glycerol. The probe 5'-AGTTGAGGGGACTTTCCCAGGC-S' (SEQ ID NO: 13) (Santa Cruz Biotechnology,

Inc., Santa Cruz, CA) was used as an NF-κB competitor, and the cells were pre-treated 50 times so as to conduct the EMSA. An NF-κB antibody supershift assay was

conducted by reacting the pre-treated cells with 1 ug of NF-κB antibodies at 4 ^°C for 30 minutes, and then the EMSA was carried out. In Fig. 8, it was seen from the EMSA

that the NF-κB was activated by the MB-ODN4/5#31 and the MB-ODN4/5#31.14 in the RAW 264.7 cells. It was confirmed from the EMSA that the MB-ODN4/5#31 and the

MB-ODN4/5#31.14 according to the present invention activate the NF-κB regardless of

the backbone shapes (both of O-type and S-type).

Induction of Humoral Immune Reaction by MB-ODN4/5#31 <5-l> Immunization

A mixture of hen egg lysozyme (HEL, 50 mg/head) and MB-ODN4/5#31 (100 ug/head) was administered intraperitoneal^ into four-week-old Balb/c mice. After one

week, a mixture of HEL and MB-ODN4/5#31 was administered at the same quantities

once again. After one week, blood was drawn using a heart punching procedure, centrifuged to obtain serum by precipitating globules. The ELISA was carried out to

measure titers of anti-HEL antibodies (the total IgG, Ig Gl, Ig G2a) from the resultant serum.

<5-2> ELISA

The resultant serum was diluted 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 a 96-well

immunoplate (Nunc), and kept at 4 ^°C for 16 hours to immobilize the HEL in the plate bottom. The plate was washed with PBST (PBS/0.05 % Tween 20), and 1 % bovine

serum albumin (BSA) was added so as to block the cells, and kept at room temperature for one hour. The serum was continuously diluted 1 :3 with PBS, sequentially added to

the plate, kept at 4 ^°C for 16 hours, and then washed with PBST. An alkaline phosphatase-conjugated detecting antibody was mixed with PBST, added to the plate, and then kept at room temperature form 2 hours. A 1 :2,000 goat anti-mouse Ig (H+L)

(Southern Biotechnology Associates) antibody was used to detect the total amount of Ig. 1-StepTM ABTS (PIERCE) was added for color fixation, and absorption was

measured at 405 nm using an ELISA reader (Labsystems) (Chu, R. S., et. al., (1997) J.

Exp. Med. 186, 1623-1631).

The MB-ODN4/5#31 was administered intraperitoneal Iy into the Balb/c mice together with hen egg lysozyme (HEL) to examine a humoral immune reaction. It was

confirmed that the MB-ODN4/5#31 segment has an adjuvant effect in the humoral

immune reaction since the level of the antibody was more increased in the mice

administered with HEL along with the MB-ODN4/5#31 , compared to the mice administered with HEL alone (Fig. 9). Freund's adjuvant, which is a reagent

manufactured by mixing an extract of Mycobacteria with paraffin oil, has been used as

one of the representative adjuvants for about 60 years. However, the adjuvant has

problems that it does not show a cell-mediated immunostimulatory effect and it should not be used in human. It was found that the MB-ODN4/5#31 could be used as a novel

adjuvant since it acts as the adjuvant for stimulating the humoral immune reaction, and

also stimulates the immune cells to induce the cell-mediated immune reaction. Also, it was shown that the MB-ODN4/5#31 was effectively used for producing the antibody of

ThI immune reaction-specific IgG2a.

Induced Production of Cytokines by MB-ODN4/5#31 <6-l> Expression of Cytokine in Dendritic Cell

a) Separation of Dendritic Cell and Its Treatment with MB-ODN4/5#31 Progenitor cell was isolated from bone marrow in the thigh of four-week-old

Balb/c mice. The isolated progenitor cell was reacted with RBC lysis solution (150

mM NH₄Cl, 10 mM potassium carbonate, 0.1 mM EDTA, pH 7.4), and then harvested.

The cell was divided into 6-well plates (Nunc) at a density of 2 X 10⁶ cells/well. 10 %

FBS-containing RPMI medium, to which IL-4 and GM-CSF (Biosource) each were added, respectively, at a density of 10 ng/ml, was added to each well so as to

differentiate the progenitor cell of bone marrow into dendritic cells (Ghosh, M., J

Immunol. 170: 5625-5629, 2003). The cells were incubated at 37 ^°C in a 5 % CO₂

incubator. The cells were incubated for 6 days while changing the used medium with a fresh medium every 2 day. Then, the cells were treated with the O-type MB-ODN4/5#31 , CpG-ODN 1826, and non-CpG-ODN 2041 according to the present invention at a level of 10 μg/ml.

b) Expression of IL- 12 in Dendritic Cells

RT-PCR was carried out to measure an expression level of IL- 12 in the dendritic

cells treated with the O-type MB-ODN4/5#31 according to the present invention.

First, the dendritic cells separated from the Balb/c mouse in the Example <6-la>

were treated with O-type MB-ODN4/5#31 at a certain time (0, 0.5, 1 , 2, 4 and 8 hours). The control groups were treated with O-type 1826 CpG ODN and 2041 non-CpG ODN,

respectively. Subsequently, the total RNA was isolated from the dendritic cells using TRIzol

(Invitrogen). Then, the total RNA (5 μg) was treated with M-MLV

reverse-transcriptase (Invitrogen) to construct cDNA. The resultant cDNA was used as the template, and a following specific primer set was used to carry out the PCR.

Forward primer (SEQ ID NO: 14) 5'-CTGGTGCAAAGAAACATGG-S' Reverse primer (SEQ ID NO: 15) 5'-TGGTTTGATGATGTCCCTGA-S'

PCR amplification was carried out by repeating 25 cycles of DNA denaturation

at 95 ^°C for 30 seconds; annealing of primers at 57 ^°C for 40 seconds and its extension

at 72 ^°C for one minute. After the PCR amplification was completed, the amplified PCR product was confirmed in the 1 % agarose gel. As a result, it was revealed that

the expression of the IL- 12 was induced only by the O-type MB-ODN4/5#31 of the

present invention, as shown in Fig. 10. Meanwhile, the expression of the IL- 12 was not induced by the S-type 1826 CpG ODN, in the contrary to the reports that the

expression of the IL- 12 was highly induced by the S-type 1826 CpG ODN (Lee, KW. et al, MoI. Immunol. 41 :955-964, 2004).

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

The ELISA was carried out after immunization so as to measure an expression

level of IL-12p40 in the mouse treated with the MB-ODN4/5#31 according to the

present invention. a) Immunization

The O-type and S-type MB-ODN4/5#31 and non-CpG-ODN 2041 (100

ug/mouse) were administered intraperitoneal Iy into four-week-old Balb/c mice,

respectively. After 24 hours, blood was drawn using a heart punching procedure,

centrifuged to obtain serum by precipitating globules. b) ELISA

First, the ELISA was carried out to measure titers of the anti-IL-12p40 and anti-IL-4 antibodies in the serum isolated from the Balb/c mouse immunized with the

MB-ODN4/5#31 , as described in the Example <5-2>. The MB-ODN4/5#31 was administered intraperitoneally into the Balb/c mice to compare the production levels of IL-12p40 and IL-4. As a result, the MB-ODN4/5#31

of the present invention induced production of the IL-12p40, but did not affect the

production level of the IL-4, as shown in Fig. 11a. And, the S-type MB-ODN4/5#31 increased the production of the IL-12p40 to a higher level. Therefore, it was seen that

the MB-ODN4/5#31 of the present invention has an effect of improving the ThI immune reactivity by inducing the production of the IL-12p40.

<6-3> Expression of IL- 12 by MB-ODN4/5#31 in Mouse Spleen Immune Cell Immune cells were harvested from a spleen of the mouse, and divided into each

well at a density of 5 x 10⁵ cells/well. Then, each cell was treated with the O-type or

S-type MB-ODN4/5#31 and non-CpG-ODN 2041 (0 or 10 μg/ml), and incubated for 24

hours. The cell culture was separated after the incubation was completed. In order to

measure a level of the cytokine in the cell culture, a sandwich ELISA was then carried out using each of the commercially available anti-IL-12 p40 and IL-4 antibodies (R&D systems, Minneapolis, Minn.), as described in the Example <5-2>.

As a result, the MB-ODN4/5#31 of the present invention highly increased the expression level of the IL- 12 p40 in the spleen immune cells regardless of the backbone

shapes, as shown in Fig. l ib. But, the MB-ODN4/5#31 of the present invention did

not affect the expression of the IL-4. Especially, the representative cytokine IL- 12,

which induces the ThI immune reaction in the Thl/Th2 immune reaction, was induced

by the MB-ODN4/5#31 of the present invention, and therefore it was confirmed that the MB-ODN4/5#31 of the present invention could induce the ThI immune reaction.

In vivo Analysis to Examine Ability to Treat Atopic Dermatitis

<7-l> Application of MB-ODN4/5#31 -Containing Ointment of the Present

Invention 6 NC/Nga mice were divided into two group: an MB-ODN4/5#31 -treated group and an untreated group. The ointment (0.2 mg/head) including the resultant O-type

MB-ODN4/5#31 was applied onto a lesion site of the atopic dermatitis in the back of

the treated group of the mice every five during 2 weeks (total 4 times). Petrolatum devoid of the CpG ODN of the present invention was applied to the untreated group of the mice in the same manner as described above.

<7-2> Observation of Lesion

The lesion site of the atopic dermatitis was visually observed 5 or 7 days after application of the ointment including the MB-ODN4/5#31 of the present invention. As a result, disappearance of the skin lesions were observed in the back of the mice to

which the O-type MB-ODN4/5#31 was applied, compared to the untreated group of the

mice, as shown in Fig. 12a. Also, skins were taken from the back of the mice to

examine an efficacy in treating the atopic dermatitis using H&E staining techniques. As a result, it was confirmed that hyperkeratosis and acanthosis were significantly

reduced in the lesion site of the mice to which the O-type MB-ODN4/5#31 of the present invention was applied, and infiltration of lymphocytes in the dermis was also reduced, which shows that the atopic dermatitis was treated in the lesion site of the mice,

as shown in Fig. 12b.

<7-3> Histological Analysis a) Expression of Cytokines

1 .5 x 1.5 cm² of skins were taken 5, 7 and 14 days after application of the

ointment including the MB-ODN4/5#31 of the present invention. Then, the skins were

fixed in a 4 % formalin solution for at least 1 day. The fixed skin tissues was treated with paraffin and cut at the thickness of 5 μm. After paraffin was removed, an

experiment was carried out according to a manual of LSAB+ kit (DAKO, Denmark), as

follows. The resultant skin tissues was treated with 3 % H₂O₂ for 10 minutes. Then,

The skin tissues were blocked by adding 10 % normal goat serum diluted with TBS

(Tris-buffered saline, pH7.4) including 0.1 % BSA. Then, the skin tissues were treated

with primary antibodies such as a goat anti-mouse IL-IO antibody, a goat anti -mouse IL-4 antibody (Santa Cruz, USA), a rat anti-mouse IFN-antibody (Pierce, USA), and reacted at 4 C for at least 12 hours. Then, the skin tissues were reacted with biotin-labeled secondary antibody at room temperature for at least 30 minutes, and then

peroxidase-labeled streptavidin was added thereto and reacted at room temperature for about 30 minutes. A DAB Substrate chromogen system (DAKO, Denmark) was used

to stain the skin tissues, and then the stained skin tissues were observed using a

microscope.

As a result, it was revealed that expression of the IL-4 was reduced, but

expression of IFN-gamma was increased in the epidermis of the mice taken 5 days after application of the ointment including the MB-ODN4/5#31 of the present invention, as

shown in Fig. 13. Therefore, it was seen that the O-type MB-ODN4/5#31 of the present invention suppresses production of the cytokine IL-4 mediated by Th2 phenotype T lymphocyte which is specifically high in the atopic dermatitis, while the O-type MB-ODN4/5#31 of the present invention improves and treats the conditions of

the atopic dermatitis by increasing the production of the cytokine IFN-gamma mediated

by ThI phenotype T lymphocyte. b) Measurement of Cell Numbers of CD4+ and CD8+ Lymphocytes 1.5 x 1.5 cm² of skins were taken 5, 7 and 14 days after application of the

ointment including the O-type MB-ODN4/5#31 of the present invention. The obtained

skin tissues were frozen with liquid nitrogen. Then, the skin tissues were inserted into

a specimen block using a Tissue-Tek OCT compound (Sakura Finetek USA, INC.), and

cut at the thickness of 5 μm using a cryostat. The cut skin tissues were reacted with the

primary antibodies such as a rat anti-mouse CD4 mAb (BD phamingen, USA) or a rat anti-CD8 mAb (Serotec, UK) at 4 ^°C for 12 hours. Then, the resultant skin tissues were reacted with biotin-labeled secondary antibody at room temperature for at least 30

minutes, and then peroxidase-labeled streptavidin was added thereto and reacted at room

temperature for about 30 minutes. A DAB Substrate chromogen system (DAKO,

Denmark) was used to stain the skin tissues, and then the stained skin tissues were observed using a microscope. The photographs all were taken at 100 magnifications.

As a result, it was revealed that the CD4+ and CD8+ lymphocytes were reduced in the skins of the mice to which the O-type MB-ODN4/5#31 of the present invention

was applied, as shown in Fig. 14. It was shown that reduction of the CD4+ and CD8+

lymphocytes in the lesion of the atopic dermatitis makes it very effective to treat the atopic dermatitis (Christian V., et al. J Clin Invest. 104:1097-1105, 1999).

<7-4> Analysis of IgE Level in Serum

Blood plasma was taken from each group of the mice, and stored at -20 ^°C until its use. The total IgE level was measured using a mouse IgE BD OptEIA Kit (BD

Phamingen, USA). In order to examine a level of IgE antibody (BD Pharmingen,

USA) in the plasma, a commercially available biotin-labeled IgE antibody (BD

pharmingen, USA) was then used to carry out a sandwich ELISA, as described above in Example <5-2>.

As a result, the IgE level in the serum was significantly reduced in the mice to

which the ointment including the O-type MB-ODN4/5#31 of the present invention was applied, as shown in Fig. 15.

From the above result, it was seen that the O-type MB-ODN4/5#31 of the

present invention increases expression of the cytokine mediated by ThI lymphocyte, while the O-type MB-ODN4/5#31 of the present invention has a very excellent efficacy

in treating the atopic dermatitis by suppressing expression of the cytokine mediated by Th2 lymphocyte to reduce the IgE level in the serum.

Effect of MB-ODN4/5#31 on Viability of Immune Cells by Irradiation

<8-l> Expression of Bcl-xs/L by Treatment of MB-ODN4/5#31 1 x 10⁵ cells/well of RAW 264.7 cells were spread on a 6-well plate, and

incubated at 37 ^°C for 24 hours in a 5 % CO₂ incubator. Each cell was treated with the synthetic oligonucleotides at a density of 10 ug/well, and incubated at 37 ^°C for 6 hours

in a 5 % CO₂ incubator. 100 ul/well of a lysis buffer was added to homogenate the RAW 264.7 cells. Cell lysate was centrifuged to obtain a supernatant (15 ul), which was used to conduct a Western blotting assay. The resultant supernatant was treated with the antibody-goat anti-mouse Bcl-xs/L, and reacted with the peroxidase-labeled secondary antibody, and then an enhanced chemiluminescence reagent (Amersham

Pharmacia Biotech, Piscataway, NJ, USA) was used to observe the Bcl-xs/L.

As a result, it was seen that the MB-ODN4/5#31 according to the present invention functions to increase viability of the cells by stimulating expression of the Bcl-xs/L in the RAW264.7 cells, as shown in Fig. 16.

<8-2> Observation of Increased Viability of Macrophage by Treatment with

MB-ODN4/5#31

3 x 10⁴ cells/well of RAW 264.7 cells were spread on a 4-well chamber slide

(Lab-TEK Chamber slide, Nalge Nunc International, Inc), and incubated at 37 ^°C for 24

hours in a 5 % CO₂ incubator. Each cell was treated with the synthetic oligonucleotides were treated at a density of 10 ug/well for 6 hours, irradiated with a 10

Gy γ-irradiator, and then incubated at 37 ^°C for 48 hours in a 5 % CO₂ incubator. A

3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (5x, 2

ug/ml) was directly added to a medium of the incubated RAW 264.7 cells (to final concentration of 0.4ug/ml), and reacted at 37 ^°C for 4 hours in a 5 % CO₂ incubator. After the media were completely removed from each well, 0.5 ml of DMSO was added,

and then reacted at 37 ^°C for 10 minutes to dissolve resultant formazan crystals. 100 ul

of a reaction solution was taken and used to measure its absorption at 570 nm.

As a result, it was seen that treatment of the MB-ODN4/5#31 according to the present invention prevents the RAW 264.7 cells from being killed by irradiation, as

shown in Fig. 17. For the backbone shapes, the O-type MB-ODN4/5#31 has the higher activity. <8-3> Observation of Increased Viability of B Cells by Treatment with

MB-ODN4/5#31

1 x 10⁵ cells/well of RPMI 8226 cells were spread on a 6-well plate, and treated

with the synthetic oligonucleotides at a density of 10 ug/well for 6 hours, irradiated with a 10 Gy γ-irradiator, and then incubated at 37 ^°C for 48 hours in a 5 % CO₂ incubator.

50 ug/ml of propidium iodide (PI) was added to the incubated cells, reacted in ice for 10

minutes, and then a level of the cell stained with PI was measured using a Flow

Cytometry.

Also, the incubated cells were washed twice with cold PBS, and 5 ul of Annexin

V-PE was added, and then reacted at room temperature for 15 minutes. 0.4 ml of an

Annexin V binding buffer was added thereto to measure a level of the cells bound to

Annexin V, using a Flow Cytometry.

As a result, it was seen that treatment of the MB-ODN4/5#31 according to the

present invention prevents the RPMI 8226 cells from being killed by irradiation, as shown in Figs. 18 and 19.

From the results described above, it was confirmed that the MB-ODN4/5#31 of the present invention has the very excellent efficacy in normalizing the immune

functions by increasing viability of the normal immune cells when the intractable

diseases such as a cancer, etc. are treated by irradiation.

INDUSTRIAL APPLICABILITY

As described above, it was seen that Mycobacterium bovis BCG-derived

oligonucleotide segments according to the present invention was involved in the humoral immune reaction by acting as the adjuvant to form the HEL antibody, and

involved in the activation of the innate immune cells by activating the IL-8 promoter in

the activation cascade of the IL-8 and IL- 12 promoters of the macrophage. Also, it was

confirmed that the oligonucleotides of the present invention might be used as a novel adjuvant since it acts as the adjuvant for stimulating the humoral immune reaction, and

also stimulates the immune cells to induce the cell-mediated immune reaction. And it was revealed that the MB-ODN of the present invention increases expression of the

cytokine mediated by ThI lymphocyte in the NC/Nga mouse, which is an animal model for the atopic dermatitis, while the MB-ODN of the present invention has a very

excellent efficacy in treating the atopic dermatitis by suppressing expression of the

cytokine mediated by Th2 lymphocyte to reduce the IgE level in the serum.

Also, it was confirmed that the MB-ODN4/5#31 of the present invention has the very excellent efficacy in normalizing the immune functions by increasing viability of

the normal immune cells when the intractable diseases such as a cancer, etc. are treated

by irradiation.

Claims

What is claimed is:

1. Oligodeoxynucleotides for stimulating an adjuvant or treating

immune-related diseases, presented in General Formula:

[General Formula] : HKCGTTCRTGTCSGM (SEQ ID NO: 1 ) wherein, R represents A or G; S represents C or G; H represents A, T or C; K

represents G or T; and M represents C or A.

2. The oligodeoxynucleotides for stimulating an adjuvant or treating immune-related diseases according to the claim 1 ,

wherein the oligonucleotides further comprise five nucleotides, presented in

following General Formula, at a 5 '-terminal end and a 3 '-terminal end:

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

wherein, R represents A or G; S represents C or G; H represents A, T or C; K

represents G or T; D represents A, G or T; M represents C or A; M represents C or A;

and Y represents C or T.

3. The oligodeoxynucleotides according to any of the claims 1 and 2, wherein the oligodeoxynucleotides protect normal immune cells when the radiotherapy is applied.

4. The oligodeoxynucleotides according to any of the claims 1 and 2,

wherein the oligodeoxynucleotides treat or prevent skin diseases.

5. The oligodeoxynucleotides according to any of the claims 1 and 2,

wherein the oligodeoxynucleotides maintain balances of various Thl/Th2 immune reactions.

6. The oligodeoxynucleotides according to any of the claims 1 and 2, wherein the oligodeoxynucleotides have a phosphodiester bond or phosphorothioate bond between the nucleotides.

7. The oligodeoxynucleotides according to any of the claims 1 and 2,

wherein the oligonucleotides are selected from the group consisting of

5'-AGCAGCGTTCGTGTCGGCCT-S ' (SEQ ID NO: 3),

5'-AGCAGCGTTCGTGTGCGCCT-S ' (SEQ ID NO: 4),

5'-AGCAGCGTTCATGTCGGCCT-S ' (SEQ ID NO: 5), 5 '-AGCAGCGTTCGTGTCCGCCT-S ' (SEQ ID NO: 6),

5'-GTATTCGTTCGTGTCGTCCT-S' (SEQ ID NO: 7) and

5 '-TGACTCGTTCGTGTCGCATG-S ' (SEQ ID NO: 8).