Title of Invention

"OLIGODEOXYNUCLEOTIDES USEFUL IN MANIPULATING AN IMMUNE REACTION"

Abstract The present invention relates to Oligodeoxynucleotides useful in manipulating an immune reaction consisting of 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.
Full Text TECHNICAL FIELD
The present invention relates to oligonucleotides derived from Mycobacterium for manipulating immune reactions, which may stimulate immune reactions and maintai n a balance of the immune reactions and have an effect on various immune-related disea ses 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, winch have an efficacy that is varied according to modification of the DNA sequences), and may be used in treatment of various immune-related diseases and an atopic dermati tis by stimulating immune reactions (an adjuvant) by the oligodeoxynucleotides in the fo rm 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 (Thl) and T helper type 2 (Th2) immunities, and between inflammation and unresponsiveness should be necessarily controlled elaboratel y. Unfortunately, conditions such as autoimmune-related diseases, allergic diseases, ch ronic inflammation, etc. have, however, been spread since many therapeutic agents for t
he immune-related diseases developed up to now could not control the immune system a dequately. However, the innate immune system is a mechanism in which immune cells
are activated by recognizing a structural difference of a foreign substance (Pathogen-A ssociated Molecular Pattern, PAMP) when a pathogen was invaded, and subsequent sign als are transmitted to initiate a cascade reaction of the immune system, resulting in destr uction of the pathogen. Accordingly, therapeutic agents of the immune-related disease s should be necessarily developed to minimize the Evil mechanism after exact understan ding of a Good & Evil mechanism using the innate immune system.
In the 1890's, William B. Coley observed a surprising result that infection of pat hogenic microorganisms may induce an anti-cancer effect in cancer patients, and therefo re it was found that its modified bacterial therapy has about 40 % of the therapeutic effe ct 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 mat an active fraction of B acillus 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. I n 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 D NA 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 character istics 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 foe used 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 immunol ogical 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 activa tion, and certain immune responses to each antigen (6, 7). An CpG dinucleotide comp osed of two nucleic acid sequences is a gist of the immunological activation and its cont rol, 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 oligodeoxynucleonde inclu ding the CpG motifs (CpG-oligodeoxynucleotide, CpG-ODN) activates various defense mechanisms of the host including innate immune responses and acquired immune respo nses (Akdis, CA. Curr Opin Jmmunol.,\2:64\-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 ba ckbone, referred to as a basic backbone of DNA, was easily decomposed in the body sin ce it was sensitive to nucleases. Accordingly, the CpG-ODN has a low risk of inducin g 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. e t al, Biochem. Biophys. Res. Commun., 311:129-138, 2003). On the other hand, the C pG-ODN with the phosphorothioate backbone was artificially engineered by modifying i ts structure so that it cannot be decomposed in vivo by the nuclease. The CpG-ODN w ith 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 wid ely used lately. However, such a CpG-ODN with the phosphorothioate backbone indu ces toxicity since it increase binding by the ODN non-specific to many proteins, and the refore it is not easily decomposed in vivo. Also, it was reported that the CpG-ODN wit h the phosphorothioate backbone induces the arthritis and exacerbates its symptoms (De ng GM et al., Arthritis & Rheumatism, 43 (2): 356-364, 2000), and causes the autoimmu ne-related diseases such as SLE (systemic lupus erythematosis) (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 vacc ine since the event of this century. However, aluminum salt (alum, AlzOs) 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 t o a mouse than when only the alum was used as the adjuvant (Davis H L. et al, J. Immu nol. 160: 870-876,1998). It was seen that the alum slightly induces cell-mediated imm unity by inducing the Th2 immune reaction, while the CpG ODN strongly induces humo ral and cell-mediated immunity by inducing expression of the Thl cytokines. However , the problem is that the CpG-ODN used hi 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 sk in of the animals including human. Amongst them, an atopic dermatitis has characteris
tic 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. et al.. Lancet. 351:1715-1721, 1998). Generally, the atopic dermatitis tends to be inherite d, and accompanied by an allergic asthma, an allergic rhinitis, an allergic conjunctivitis and an urticaria, depending on individuals. A series of immunological abnormalities re ported in the atopic dermatitis patients include an increased production of IgE, the reduc ed number and deteriorated function of CD8+ suppressor/cytotoxic T lymphocytes, the r educed number of Thl (T-cell Helper type 1) lymphocyte that secretes IFN-gamma, etc. Also, T lymphocyte having histological CD4+ phenotype, infiltration of monocytes/m acrophages, mast cells and eosinophils are increased in the skin abnormality of the atopi c 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. Dermato L, 96:523-526,1991).
Many researchers have developed the methods for treating the cancer by killing t he 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 irradia tion, resulting in its reduced immune functions. It has been reported that the immune c ells such as B cells (Ashwell JD et al., J. Immunol 136:3649-3656, 1986), T cells (Pros ser JS Int. J. Radial. Biol. Relat. Stud. Phys. Chem. Med. 30:459-465,1976), macrophag es (Yoshihisa K et al., J. Radiat Res. 45:205-211, 2004), etc. were killed by the irradiati on (apoptosis). Accordingly, among the radiotherapeutic methods for treating the disea ses such as a cancer, etc., there are required the methods that normal immune cells exce pt the cancerous cells are survived to normally maintain the immune reactions.
The present invention relates to oligonucleotides derived from Mycobacterium b ovis BCG for manipulating immune reactions, which may be used in treatment of varkm s immune-related diseases by stimulating immune reactions (an adjuvant) and maintaini ng a balance of the immune reactions, and also have effects of treating an atopic dermati tis 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 Ge
neral Formula and composed of DNA sequences including at least two unmetbylated Cp
G motifs, wherein Ifae CpG oligodeoxynucleotides may be used to stimulate immune rea
ctions (an adjuvant), maintain a balance of Thl/Th2 immune reactions so as to treat van
ous immune-related diseases, and protect normal immune cells when intractable disease
s 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 rep resents G or T; and M represents C or A.
In the present invention, the oligonucleotides preferably further include five nucl eotides, 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 rep
resents 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 inclu des 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 (hereinafte r, 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 a n animal including human. The subject may be a patient in need of treatment
In the present invention, the oligonucleotides is preferably selected from the grou p consisting of 5'-AGCAGCGTTCGTGTCGGCCT-3' (SEQ ID NO: 3), 5'-AGCAGCG TTCGTGTGCGCCT-3' (SEQ BDNO: 4), 5'-AGCAGCGTTCATGTCGGCCT-3' (SEQ ID NO: 5), 5'-AGCAGCGTTCGTGTCCGCCT-3' (SEQ ID NO: 6), 5'-GTATTCGTT CGTGTCGTCCT-3' (SEQ ID NO: 7), and 5'-TGACTCGTTCGTGTCGCATG-3' (SE Q ID NO: 8).
The MB-ODN of the present invention may be derived from natural sources (for example, chromosomal DNA ofM. bovis BCG), and chemically synthesized, or recombi nantly 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 Interscienc e, 1989); Maniatis, et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbo r Lab., New York, 1982); and U.S Patent No. 4,458,066).
The MB-ODN of the present invention preferably has a phosphodiester backbon e. The phosphodiester backbone, referred to as a basic backbone of DNA, has a low ris k of inducing in vivo toxicity since it is easily decomposed in vivo by the nucleases. Th e MB-ODN of the present invention is characterized in that it has an excellent immunol ogical 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 in elude modified backbones. It was revealed that modification of the oligonucleotide ba ckbone might allow the CpG ODN to strengthen the activity and/or stability when the C pG-ODN is administered in vivo. In the MB-ODN of the present invention, the preferr ed modification of the backbone includes modification into phosphorothioate, which is a Howed to be resistant to its decomposition. The modification into phosphorothioate m ay be generated at the terminal ends, and for example two or three of the last 5' or 3' nu cleotides may be connected by phosphorothioate bonds. Also, the MB-ODN of the pre sent 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 oft he present invention may be modified to have a partially phosphorothioate-modified bac kbone. The phosphorothioate may be synthesized by the automatic techniques using p hosphoramidate or H-phosphonate chemistry (S. E. Beaucage et al., Tetrahedron Lett., 2 2:1859, 1981; Froehler et al., Nucl. Acid. Res., 14:5399-5407). As another modificatio n 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 alkyl ated, as described in U.S. Patent No. 5,023,243 and EP Patent No. 092,574) may be man ufactured by an automatic solid-phase synthesis using commercially available reagents.
Also, still another modification example, which makes the MB-ODN less sensitive to t he decomposition, includes acetyl-, thio- and similar modifications of adenosine, cytosin e, 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 terrain al ends is also more resistant to its decomposition, hi addition, the CpG-ODN further i ncludes combination of phosphodiester and phosphorothioate, phosphotriester, phospho ramidate, methylphosphonate, methylphosphorothionate, phosphorodithoate and combin ations 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 backb one may have stronger immunological effects by means of enhanced nuclease resistance , increased cellular uptake, increased protein uptake and/or altered intracellular localizati on, etc.
The preferred backbone of the MB-ODN of the present invention is a phosphodi ester (hereinafter, referred to as "O-type") or phosphorothioate (hereinafter, referred to a s "S-type") backbone, and the most preferred backbone is the O-type backbone that is n ot easily decomposed in vivo to induce side effects.
It was seen that the MB-ODN according to the present invention strongly induce s the humoral immune reactions by inducing expression of the Thl cytokines, and has a n adjuvant activity that improves efficiency of the vaccine. Specific physiological acti vities are as follows:
1) Production of EL-12 is increased in immune cells from a mouse and a mouse s
pleen.
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 a s an antigen and an adjuvant, respectively. At this time, it is revealed that production o f IgG2a is more increased as a result of the Thl immune reaction when CFA is used as a n 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 con ventional CpG-ODNs known in the prior art, the MB-ODN of the present invention is c haracterized 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 modi fied into an O-type backbone has nearly the same activity as the CpG-ODN modified int o an S-tvpe 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 hu moral immune reactions by inducing expression of the Thl cytokines.
The MB-ODN according to the present invention has the physiological activities that control balance of the Thl/Tn2 immune reaction by suppressing the Thl cytokine (f or example, IL-4), and/or inducing the Thl cytokine (for example, IL-12). Specific ph ysiologjcal activities are as follows: 1) Macrophages are activated to activate an IL-12 pr omoter; 2) Dendritic cells are activated to induce expression of the DL-12; 3) Production of the EL-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 (EL-4 and IL-10) mediated by Th2-ly mphocytes 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. U nlike the conventional CpG-ODNs known in the prior art, the CpG-ODN of the present i nvention is characterized in that it has nearly the same activity regardless of its backbon e shape. In the present invention, it was revealed that the CpG-ODN of the present inv ention modified into an O-type backbone has nearly the same activity as, or the more ex cellent 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 unbala nee of the Thl/Th2 immune reaction since the balance of theThl/Th2 immune reaction i s maintained by inducing expression of the Thl cytokines.
The MB-ODN according to the present invention has an effect of increasing viab ility of the immune cells. The MB-ODN has effects of stimulating macrophages to inc rease 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 t
he immune functions by increasing the viability of the normal immune cells when intrac
table diseases such as a cancer, etc. are treated using the irradiation. Specific physiolo
gical 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; an
d 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 t he present invention will be more fully described in the following detailed description, t aken accompanying drawings, incorporated herein in its entirety by this reference. In t he drawings:
Fig. 1 is a result of analyzing chromosomal DNA sequences of E. coli and M. bo
vis BCG using a computer program. All DNA sequences (CpG motifs) in which two b
ases 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 chromoso
mal DNA of M bovis BCG, as shown hi Fig. 1.
Fig. 2 is a result of analyzing DNA sequences in which three CpG motifs are pre sent on 20 base pairs among the DNA sequences present in the chromosomal DNA of M . bovis BCG. hi 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 b etween the bases C and C (-CGXXXCGXXXCG-, MB-ODN 5/5). It is shown that 39 5 oligonucleotides in the form of-CGXXCGXXXCG- and 354 oligonucleotides in the f orm 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 i mmune reactions are selected and synthesized, and the used for detecting the candidate s equences.
Fig. 4 is a diagram showing activation of EL-8 and EL-12 promoters in RAW 264. 7 cells treated with the 71 oligonucleotides for controlling the immune reactions, synthe sized in the form of the phosphodiester bond as shown in Fig. 3. Fig. 4a is a diagram s howing a result of comparing how much 35 synthesized oligonucleotides in the MB-OD
N 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 fo rm activate an IL-12 promoter of the macrophage, and Fig. 4c is a diagram showing a re suit of comparing how much 35 synthesized oligonucleotides in 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 CGTTCGTGTC G of MB-ODN 4/5#31 present on 20 base pairs among the DNA sequences present in th e chromosomal DNA of M. bovis BCG (Fig. 5a), and then synthesizing the oligonucleoti des with the phosphodiester backbones to compare how much the 17 oligonucleotides a ctivate an IL-8 promoter of the macrophage (Fig. 5b).
Fig. 6 is a diagram showing a result of comparing how much the oligonucleoti de s 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 hi 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 EL-12 promoters in a mouse macrophage ce 11 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 ac tivate the IL-8 promoter in a concentration-dependant manner. Figs. 7c and d are diagr ams 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 DL-8 and EL-12 promoters.
Fig. 8 is a diagram showing that NF-xfi is activated when the RAW 264.7 cell li nes 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-KB is confirmed by treating and fixing the RAW 264.7 cells with MB-ODN 4/5 #31(10 ug/ml) for 30 minutes, followed by conducting an indirect immunofluorescence assay using the NF-xB p65-specific antisera. Fig. 8b shows a result of an electrophoret ic mobility shift assay (EMSA) in which the RAW 264.7 cells are treated with MB-OD N 4/5#31(10 ug/ml) for 30 minutes, and then nucleoproteins are isolated to confirm bin ding of the NF-xB to a NF-KB consensus binding site.
Fig. 9 is a diagram showing that the backbones of the MB-ODN 4/5#31 are synt hesized 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 ly sozyme (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 a nd non-CpG-ODN (2041) for expression of the EL-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 convention al 1826 CpG-ODN and non-CpG-ODN (2041) for production of the EL-12 p40. Fig. 1 la is a diagram confirming how much the EL-12 p40 is produced in the blood serum afte r the Balb/c mice is abdominally immunized with the MB-ODN 4/5#31. Fig. 11 a is a d
iagram confirming a level of the produced IL-12 p40 when spleen immune cells is separ ated 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 administr ation of the O-type MB-ODN 4/5#31 according to the present invention using the anima 1 model.
Fig. 12a is a photograph showing that a NC/Nga mouse is examined with the nak ed eye on 5 and 7 days after the O-type MB-ODN 4/5#31 of the present invention is app lied to an atopic dermatitis lesion present in the back of the NC/Nga mouse; and Fig. 12 b is a photograph showing that the O-type MB-ODN 4/5#31 is applied to the dorsal skin of tiie NC/Nga mouse in which the atopic dermatitis is broken out, extracted out and th en 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 w here a level of the expressed cytokines (IL-4 and EFN-gamma) is observed in the dorsal s kin of the NC/Nga mouse to which the O-type MB-ODN 4/5#31 of the present inventio n 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 w here the cell number of the CD4+ and CD8+ lymphocytes is observed hi 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 admini stered. In the drawing, "AD" represents an untreated group.
Fig. 1 6 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 RAW264.7 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 viab ility of the RPMI 8226 cells is increased when the B cell line RPMI 8226 is irradiated w ith the radiation after the RPMI 8226 cells are pre-treated with the MB-ODN 4/5#31 .
Fig. 1 9 is a diagram showing, using an flow cytometry after Annexin V staining, mat viability of the RPMI 8226 cells is increased when the B cell line RPMI 8226 is irra dialed with the radiation after the RPMI 8226 cells are pre-treated with the MB-ODN 4/
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 pu rpose 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
Analysis of DNA Sequences of CpG Motifs in the Chromosomal DNAs fr om 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 cal culated using the computer program. It was found that the probability of the DNA seq uence XXCGXX on the chromosomal DNA is theoretically 1/46, but the probability oft he sequence XXCGXX in the chromosomal DNAs of E. coli and M. bovis BCG is actua lly much higher. Also, it was confirmed that frequency of the sequence XXCGXX in t he chromosomal DNA of M bovis BCG is more higher than that of E. coli (Fig. 1).
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 ch romosomal DNA, and then the oligonucleotides including the three motifs XXCGXX w ere 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 -CG XXCGXXXCG- and 354 oligonucleotides in the form of-CGXXXCGXXXCG- are pre sent 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 includin
g the high frequencies of the motif XXCGXX, as shown in Fig. 1. The oligonucleotide s whose CG is present in the 5'- or 3'- terminal end of 20 base pairs of the oligonucleoti des was excluded, and then the 71 candidate oligonucleotides for controlling the immun e reaction were selected, synthesized and used for detecting the candidate substances.

Detection of MB-ODN Having Immune Activity
Immune Reaction of Synthesized Candidate MB-ODNs
It was examined whether or not the MB-ODNs prepared in the Example , a
nd their various substituents could activate the IL-8 and IL-12 promoters of the macroph
ages.
a) Cultivation of Mouse Macrophage
Raw 264.7 cells (ATCC, Manassas, VA) were cultured in a DMEM medium incl uding 10 % FBS (Gibco BRL). Cell culture was carried out in a 5 % CO2 incubator (F orma) at 37 "C.
b) Design of IL-8 and IL-12 promoter-Iuc Reporter Plasmid
In order to amplify an IL-8 promoter region (from -135 bp to +46 bp), human ge nome DNA was used as a template, and following primer sets were used to conduct a P CR reaction.

A fragment of the amplified IL-8 promoter region was inserted into a pGL3-Basi c plasmid (Promega) digested by the restriction enzymes BglG. and HindlTL. Therefore,
an DL-8 promoter-Zwc 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 b p), human genome DNA was used as a template, and following primer sets were used to
conduct a PCR reaction.

A fragment of the amplified EL-12 promoter region was inserted into a pGL3-Ba sic plasmid (Promega) digested by the restriction enzymes Sac I and Xho I. Therefore, an IL-12 promoter-Ii/c 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, MO) were divided into 12-well plates at a
concentration of 5X104 cells/well and cultured at 37 °C for 24 hours in a 5 % COa inc ubator. The cells were co-transfected with the EL-8 promoter-Zwc reporter plasmid or t he IL-12 promoter-Z,wc reporter plasmid, which were constructed in the b), and a pRL-n ull plasmid (Promega). Then, the co-transfected cells were cultured at 37 °C for 24 h ours in the 5 % CO2 incubator. Each well was treated with the MB-ODNs (10 ug/well)
shown in the Fig. 3, and cultured at 37 "C for 6 hours or 12 hours in the 5 % CO2 incu bator. 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 ul/well to homogenize the cells. The cell lysates was centrifuge
d, and the resultant supernatant (15 ul) was used to conduct a luciferase assay. The luc iferase activity was measured using a TD-20/20 (Turner designs) luminometer. Each p remoter activity by treatment of the MB-ODNs was measured as a relative activity of th e control group. That is, if activity of the control group was set to ' 1', 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 acti vates the IL-8 promoter, as shown in Fig. 4.
Activation of IL-8 Promoter by Oligonucleotides Homologous to MB-OD N4/5#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 oligonuc leotides having different DNA sequences except that they have the sequence CGTTCGT GTCG within fee DNA sequences of MB-ODN4/5#3l having the effect of the IL-8 pro moter activation, as shown in the Example . As a result, it was seen that 17 oligo nucleotides homologous to the MB-ODN4/5#31 are present, as shown in Fig. 5a. And then, the same method as in the Example was repeated to measure the BL-8 promo ter activity.
Accordingly, it was revealed that the ability to activate the EL-8 promoter is varie d depending on the DNA sequences, as shown in Fig. 5b. It was seen that the MB-OD N4/5#31.14 also has high activity, in addition to the MB-ODN4/5#31 according to the p resent invention.

Modification and Immune Reaction of the DNA Sequence of the Oligonucleotid eMB-ODN4/5#31
Modification of DNA Sequence of the Oligonucleotide MB-ODN4/5#31
The DNA sequence of the oligonucleotide MB-ODN4/5#31 was modified to syn thesize DNA sequences, as described Fig. 6. Each CG sequence of the MB-ODN4/5#3 1 was changed to a GC sequence (#31-CG-1, #31-CG-2, #31-CG-3). Also, the first C G and the second CG were changed to a GC sequence (#3 l-CG-4), the second CG and t he 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 w as 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 s equence C A, and the first and third CG was changed to the sequence CA.
Measurement of Immune Reactions by Oligonucleotides Modified from t he Oligonucleotide MB-ODN4/5#31
5xl04 cells/well of RAW 264.7 cells were spread on a 12-well plate, and incubat ed at 37 1C for 24 hours in a 5 % C02 incubator. An IL-8 promoter reporter plasmid a nd a pRL-null plasmid were co-transfected, and then incubated at 37 t! for 24 hours in a 5 % CO2 incubator. Each well was treated with synthetic oligonucleotides at 10 ug/w ell, and incubated at 37 °C for 6 hours hi a 5 % COi incubator. Then, the same method as in the Example was repeated to measure the IL-8 promoter activities.
The luciferase assay was used for measuring how much the synthetic oligonucle otides having any modified DNA sequences activates the IL-8 promoter of the macropha
ge. As a result, the IL-8 promoter was highly activated by the oligonucleotides 5'-AG CAGCGTTCGTGTGCGCCT-3', 5'-AGCAGCGTTCATGTCGGCCT-3' 5'-AGCAGC GTTCGTGTCCGCCT-3' (Fig. 6b). Other synthetic oligonucleotides showed lower IL -8 promoter activities than the control group. In the oligonucleotides that activate the I L-8 promoter, the DL-8 promoter activities were measured even by the oligonucleotide h aving 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 sequenc es GTGCGC and GTCCGC reappearing in the CpG motif could activate the IL-8 promo ter(Fig.6).

Examination of Immune Reaction by Backbone Modifications of Oligonucleotid esMB-ODN4/5#31 andMB-ODN4/S#3U4
Activation of RAW 264.7 Cells by Backbone Modifications of MB-ODN 4/5#31 and MB-ODN4/5#31.14
RAW 264.7 cells were co-transfected with IL-8-Z,uc promoter reporter vector or IL-12-Z,wc promoter reporter vector constructed in the step b) of the Example , and pRL-null plasmid (Promega). The transfected cells were treated with the O-type (pho sphodiester backbone) and S-type (phosphorothioate backbone) MB-ODN4/5#31 and M B-ODN4/5#31.14 (0 or 10 ug/ml), and incubated for 8 hours, respectively. Then, the s ame method as in the Example was repeated to measure activities of the IL-8 pro moter and the IL-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 reg
ardless of the backbone shapes (both of O-type and S-type), as shown in Fig. 7.
Activation of NF-KB 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 a t a concentration of 5 x 105 cells/ml and incubated at 37 *C for 24 hours in a 5 % COi in :ubator. Each well was treated with the oligonucleotides MB-ODN4/5#31 and MB-O DN4/5#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 ant i-p65 (titer 1: SOO) antibodies was added to PBST and kept at room temperature for 2 ho urs. After the cells were washed with PBST, they were treated with Donkey-anti-mous j-IgG-FTTC (titer 1:250) antibodies for 2 hours. Mobilization of the NF-icB into nuclei was observed using a confbcal microscopy (Lee, Y., et. al., (2002) Blood 99,4307-431
7)-
Fig. 8a is a photograph showing that NF-icB was stained using an immunostainin
I method and mobilization of the NF-icB into nuclei was observed using the confocal mi
;roscopy. The NF-xB 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-KB was mobilized into t
ic nuclei. The oligonucleotides MB-ODN4/5#31 and MB-ODN4/5#31.14 according t
) 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-icB is activated in RAW 2 64.7 cell lines treated with MB-ODN4/5#31 and MB-ODN4/5#31.14 using an electroph oretic mobility shift assay (EMSA). 5 x 105 cells/ml of RAW 264.7 cells were added t o 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-3') (SEQ ID NO: 13), wh ich has a NF-icB binding site, was labeled with 32P for the EMSA. The 32P-labeled pro be and 20 ug of the nucleoprotein were mixed in a buffer (10 mM HEPES, pH 7.9,65 m M NaCl, 1 mM dithiothreitol, 0.2 mM EDTA, 0.02 % NP-40, 50 mg/ml poly (dldC)rpol y (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 prob e S'-AGTTGAGGGGACTTTCCCAGGC-S' (SEQ ID NO: 13) (Santa Cruz Biotechnolo gy, Inc., Santa Cruz, CA) was used as an NF-KB competitor, and the cells were pre-treat ed 50 times so as to conduct the EMSA. An NF-icB antibody supershift assay was con ducted by reacting the pre-treated cells with 1 ug of NF-icB antibodies at 4 °C for 30 min utes, and then the EMSA was carried out. hi Fig. 8, it was seen from the EMSA that th e NF-KB 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-icB regardless of the b ackbone shapes (both of O-type and S-type).

Induction of Humoral Immune Reaction by MB-ODN4/5#31
Immunization
A mixture of hen egg lysozyme (HEL, 50 mg/head) and MB-ODN4/5#31 (100 u g/head) was administered intraperitoneally 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 o nee again. After one week, blood was drawn using a heart punching procedure, centrif uged to obtain serum by precipitating globules. The ELISA was carried out to measure liters of anti-HEL antibodies (the total IgG, Ig Gl, Ig G2a) from the resultant serum.
ELISA
The resultant serum was diluted 1:10 using PBS/0.2 % sodium azide, and stored
at -20 t. HEL (10 ug/ml sodium bicarbonate buffer, pH 9.6) was added to a 96-well i
mmunoplate (Nunc), and kept at 4 "C for 16 hours to immobilize the HEL in the plate b
ottom. The plate was washed with PBST (PBS/0.05 % Tween 20), and 1 % bovine ser
urn 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 phosphatas
e-conjugated detecting antibody was mixed with PBST, added to the plate, and then kep
t at room temperature form 2 hours. A 1:2,000 goat anti-mouse Ig (H+L) (Southern Bi
otechnology 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 run
using an ELISA reader (Labsystems) (Chu, R. S., et. al., (1997) J. Exp. Med. 186,1623
-1631).
The MB-ODN4/5#31 was administered intraperitoneally into the Balb/c mice to gether with hen egg lysozyme (HEL) to examine a humoral immune reaction. It was c onfirmed that the MB-ODN4/5#31 segment has an adjuvant effect in the humoral immu ne 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 HE L alone (Fig. 9). Freund's adjuvant, which is a reagent manufactured by mixing an extr act of Mycobacteria with paraffin oil, has been used as one of the representative adjuvan ts 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 t hat the MB-ODN4/5#31 could be used as a novel adjuvant since it acts as the adjuvant f or stimulating the humoral immune reaction, and also stimulates the immune cells to ind uce the cell-mediated immune reaction. Also, it was shown that the MB-ODN4/5#31 was effectively used for producing the antibody of Thl immune reaction-specific IgG2a.

Induced Production of Cvtokines bv MB-QDN4/5#31
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 Bal b/c mice. The isolated progenitor cell was reacted with RBC lysis solution (150 mM N HjCl, 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, r espectively, at a density of 10 ng/ml, was added to each well so as to differentiate the pr ogenitor cell of bone marrow into dendritic cells (Ghosh, M., J Immunol. 170: 5625-562 9,2003). The cells were incubated at 37 t in a 5 % CO2 incubator. The cells were i ncubated 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 no n-CpG-ODN 2041 according to the present invention at a level of 10 ng/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 celk 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 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 OD N, respectively.
Subsequently, the total RNA was isolated from the dendritic cells using TRIzol ( Invitrogen). Then, the total RNA (5 ug) was treated with M-MLV reverse-transcriptas e (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.

PCR amplification was carried out by repeating 25 cycles of DNA denaturation a 195 °C for 30 seconds; annealing of primers at 57 °C for 40 seconds and its extension at 72 1C 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 exp ression of the IL-12 was induced only by the O-type MB-ODN4/5#31 of the present inv ention, as shown in Fig. 10. Meanwhile, the expression of the IL-12 was not induced b y the S-type 1826 CpG ODN, in the contrary to the reports that the expression of the EL-12 was highly induced by the S-type 1826 CpG ODN (Lee, KW. et al, Mol. Immunol. 4 1:955-964,2004).
Expression of IL-12 by MB-ODN4/5#31 hi Mouse
The ELISA was carried out after immunization so as to measure an expression le vel of IL-12p40 in the mouse treated with the MB-ODN4/5#31 according to the present invention.
The O-type and S-type MB-ODN4/5#31 and non-CpG-ODN 2041 (100 ug/mous e) were administered intraperitoneally into four-week-old Balh/c mice, respectively. A fter 24 hours, blood was drawn using a heart punching procedure, centrifuged to obtain s erum by precipitating globules.
b) ELISA
First, the ELISA was carried out to measure liters of the anti-IL-12p40 and anti-I L-4 antibodies in the serum isolated from the Balb/c mouse immunized with the MB-O DN4/5#31, as described in the Example .
The MB-ODN4/5#31 was administered intraperitoneally into the Balb/c mice to compare the production levels of EL-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 produ ction level of the EL-4, as shown hi Fig. 1 la. And, the S-type MB-ODN4/5#3 1 increas
ed 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 Thl immune reacti vity by inducing the production of the IL-12p40.
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 ug/ml), and incubated for 24 h ours. 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 o ut using each of the commercially available anti-IL-12 p40 and IL-4 antibodies (R&D sy stems, Minneapolis, Minn.)' as described in the Example .
As a result, the MB-ODN4/5#31 of the present invention highly increased the ex pression level of the IL-12 p40 in the spleen immune cells regardless of the backbone sh apes, as shown in Fig. 1 Ib. But, the MB-ODN4/5#31 of the present invention did not affect the expression of the IL-4. Especially, the representative cytokine EL-12, which i nduces the Thl 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-O DN4/5#31 of the present invention could induce the Thl immune reaction.

In vivo Analysis to Examine Ability to Treat Atopic Dermatitis
Application of MB-ODN4/5#31 -Containing Ointment of the Present Inve
ntion
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 M B-ODN4/5#31 was applied onto a lesion site of the atopic dermatitis in the back of the t reated 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.
Observation of Lesion
The lesion site of the atopic dermatitis was visually observed 5 or 7 days after ap plication 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, a s shown in Fig. 12a. Also, skins were taken from the back of the mice to examine an e fficacy in treating the atopic dermatitis using H&E staining techniques. As a result, it was confirmed that hyperkeratosis and acanthosis were significantly reduced hi the lesio n site of the mice to which the O-type MB-ODN4/5#31 of the present invention was app lied, and infiltration of lymphocytes in the dermis was also reduced, which shows that th e atopic dermatitis was treated in the lesion site of the mice, as shown in Fig. 12b.
Histological Analysis
a) Expression of Cvtokines
1.5 x 1.5 cm2 of skins were taken 5, 7 and 14 days after application of the ointme nt including the MB-ODN4/5#31 of the present invention. Then, the skins were fixed i n a 4 % formalin solution for at least 1 day. The fixed skin tissues was treated with par affin and cut at the thickness of 5 nm. After paraffin was removed, an experiment was
carried out according to a manual of LSAB+ kit (DAKO, Denmark), as follows. The r esultant 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 salin e, pH7.4) including 0.1 % BS A. Then, the skin tissues were treated with primary antib odies such as a goat anti-mouse IL-10 antibody, a goat anti-mouse EL-4 antibody (Santa Cruz, USA), a rat anti-mouse IFN-antibody (Pierce, USA), and reacted at 4 °C for at lea st 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 w as added thereto and reacted at room temperature for about 30 minutes. A DAB Substr ate chromogen system (DAKO, Denmark) was used to stain the skin tissues, and then th e stained skin tissues were observed using a microscope.
As a result, it was revealed that expression of the BL-4 was reduced, but expressi on of DFN-gamma was increased in the epidermis of the mice taken 5 days after applicati on 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 inventio n 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 incre asing the production of the cytokine IFN-gamma mediated by Thl phenotype T lymphoc yte.
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 ointme nt including the O-type MB-ODN4/5#31 of the present invention. The obtained skin ti
ssues were frozen with liquid nitrogen. Then, the skin tissues were inserted into a spec imen block using a Tissue-Tek OCT compound (Sakura Finetek USA, INC.), and cut at the thickness of 5 um using a cryostat. The cut skin tissues were reacted with the prim ary antibodies such as a rat anti-mouse CD4 mAb (BD phamingen, USA) or a rat anti-C D8 mAb (Serotec, UK) at 4 °C for 12 hours. Then, the resultant skin tissues were react ed with biotin-labeled secondary antibody at room temperature for at least 30 minutes, a nd then peroxidase-labeled streptavidin was added thereto and reacted at room temperat ure for about 30 minutes. A DAB Substrate chromogen system (DAKO, Denmark) wa s used to stain the skin tissues, and then the stained skin tissues were observed using a m icroscope. The photographs all were token 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 w as applied, as shown in Fig. 14. It was shown that reduction of the CD4+ and CD8+ ly mphocytes in the lesion of the atopic dermatitis makes it very effective to treat the atopi c dermatitis (Christian V., et al. JClin Invest. 104:1097-1105,1999).
Analysis of IgE Level in Serum
Blood plasma was taken from each group of the mice, and stored at -20 1C until i ts use. The total IgE level was measured using a mouse IgE BD OptEIA Kit (BD Pha mingen, USA), hi order to examine a level of IgE antibody (BD Pharmingen, USA) in the plasma, a commercially available biotin-labeled IgE antibody (BD pharmingen, US A) was then used to carry out a sandwich ELISA, as described above in Example .
As a result, the IgE level in the serum was significantly reduced in the mice to w hich the ointment including the O-type MB-ODN4/5#31 of the present invention was ap
plied, as shown in Fig. 15.
From the above result, it was seen that the O-type MB-ODN4/5#31 of the presen t invention increases expression of the cytokine mediated by Thl 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 lymph ocyte to reduce the IgE level in the serum.

Effect of MB-QDN4/5#31 on Viability of Immune Cells by Irradiation Expression of Bcl-xs/L by Treatment of MB-ODN4/5#31 1 x 10s cells/well of RAW 264.7 cells were spread on a 6-well plate, and incubat ed at 37 T) for 24 hours in a 5 % COa incubator. Each cell was treated with the synthet ic oligonucleotides at a density of 10 ug/well, and incubated at 37 T for 6 hours in a 5 % CQz incubator. 100 ul/well of a lysis buffer was added to homogenate the RAW 26 4.7 cells. Cell lysate was centrifuged to obtain a supernatant (15 ul), which was used t o conduct a Western blotting assay. The resultant supernatant was treated with the anti body-goat anti-mouse Bcl-xs/L, and reacted with the peroxidase-labeled secondary antib ody, 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 inventi on functions to increase viability of the cells by stimulating expression of the Bcl-xs/L i n the RAW264.7 cells, as shown in Fig. 16.
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 (La b-TEK Chamber slide, Nalge Nunc International, Inc), and incubated at 37 °C for 24 ho urs in a 5 % CC>2 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 y-irradiator, a nd then incubated at 37 °C for 48 hours in a 5 % CC*2 incubator. A 3-(4,5-dimethylthia zole-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/m 1), and reacted at 37 T) for 4 hours in a 5 % CC>2 incubator. After the media were com pletely removed from each well, 0.5 ml of DMSO was added, and then reacted at 37 1C for 10 minutes to dissolve resultant fonnazan crystals. 100 ul of a reaction solution wa s 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 pr esent 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 activ ity.
Observation of Increased Viability of B Cells by Treatment with MB-OD N4/5#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 y-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 1 0 minutes, and then a level of the cell stained with PI was measured using a Flow Cytom
etry.
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 A nnexin V binding buffer was added thereto to measure a level of the cells bound to Anne xin V, using a Flow Cytometry.
As a result, it was seen that treatment of the MB-ODN4/5#31 according to the pr esent 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 oft he present invention has the very excellent efficacy in normalising the immune function s by increasing viability of the normal immune cells when the intractable diseases such a s a cancer, etc. are treated by irradiation.
INDUSTRIAL APPLICABILITY
As described above, it was seen that Mycobacterium bovis BCG-derived oligonu cleotide segments according to the present invention was involved in the humoral immu ne reaction by acting as the adjuvant to form the HEL antibody, and involved in the acti vation of the innate immune cells by activating the IL-8 promoter in the activation casca de 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 im mune 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 Thl
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 Th.2 lymphocyt e 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 th e normal immune cells when the intractable diseases such as a cancer, etc. are treated by
irradiation.






We Claim:
1. Oligodeoxynucleotides useful in manipulating an immune reaction consisting of 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 useful in manipulating an immune reaction according to the claim 1, wherein the oligonucleotides optionally comprises five nucleotides, at a 5 '-terminal end and a 3 '-terminal end and consisting of 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 oligonucleotides are selected from the group consisting of 5'-AGCAGCGTTCGTGTCGGCCT-3 ' (SEQ ID NO: 3), 5'-AGCAGCGTTCATGTCGGCCT-3 ' (SEQ ID NO: 5) and
5 '-AGCAGCGTTCGTGTCCGCCT-3 ' (SEQ ID NO: 6),

Documents:

2871-DELNP-2006-Abstract-(07-09-2010).pdf

2871-DELNP-2006-Abstract-(22-02-2011).pdf

2871-delnp-2006-abstract.pdf

2871-DELNP-2006-Claims-(07-09-2010).pdf

2871-DELNP-2006-Claims-(22-02-2011).pdf

2871-delnp-2006-claims.pdf

2871-DELNP-2006-Correspondence-Others-(05-06-2007).pdf

2871-DELNP-2006-Correspondence-Others-(07-09-2010).pdf

2871-DELNP-2006-Correspondence-Others-(17-02-2011).pdf

2871-DELNP-2006-Correspondence-Others-(19-05-2006).pdf

2871-DELNP-2006-Correspondence-Others-(19-05-2010).pdf

2871-delnp-2006-description (complete).pdf

2871-delnp-2006-drawings.pdf

2871-DELNP-2006-Form-1-(07-09-2010).pdf

2871-delnp-2006-form-1.pdf

2871-delnp-2006-form-18.pdf

2871-DELNP-2006-Form-2-(07-09-2010).pdf

2871-delnp-2006-form-2.pdf

2871-delnp-2006-form-26.pdf

2871-DELNP-2006-Form-3-(17-02-2011).pdf

2871-DELNP-2006-Form-3-(19-05-2010).pdf

2871-delnp-2006-form-3.pdf

2871-delnp-2006-form-5.pdf

2871-delnp-2006-pct-request form.pdf

2871-DELNP-2006-Petition 137-(07-09-2010).pdf


Patent Number 246986
Indian Patent Application Number 2871/DELNP/2006
PG Journal Number 12/2011
Publication Date 25-Mar-2011
Grant Date 23-Mar-2011
Date of Filing 19-May-2006
Name of Patentee KWON, HYUNG-JOO
Applicant Address 104-1506 KUNYOUNG APT., YONGAM-DONG, SANGDANG-GU, CHEONGJU-SI, CHUNGCHEONGBUK-DO 360-770, REPUBLIC OF KOREA
Inventors:
# Inventor's Name Inventor's Address
1 KIM,TAE-YOON 8-702 ASIA SEONSUCHON APT. 86,JAMSIL 7-DONG, SONGPA-GU, SEOUL 138-797, REPUBLIC OF KOREA
2 KIM, DOO-SIK 84-3 YEONHUI 2-DONG, SEODAEMUN-GU, SEOUL 120-824, REPUBLIC OF KOREA
3 KWON, HYUNG-JOO 104-1506 KUNYOUNG APT., YONGAM-DONG, SANGDANG-GU, CHEONGJU-SI, CHUNGCHEONGBUK-DO 360-770, REPUBLIC OF KOREA
PCT International Classification Number A61K31/7088; A61P37/00
PCT International Application Number PCT/KR2005/000266
PCT International Filing date 2005-01-28
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA