Immunostimulatory Oligonucleotides

Abstract

To provide immunostimulative single-stranded oligodeoxynucleotides having excellent resistance to nucleases.SOLUTION: Disclosed is a single-stranded oligodeoxynucleotide of 14-100 deoxynucleotides, of which base sequence comprises four same polyG sites consisting of contiguous two to five guanines (Gs), where two of the polyG sites are located at 5' and 3' ends respectively, polyG sites are linked each other via a spacer between them, where the spacers comprise one or more bases, and at least one of the spacers contain one to five CpG sites comprising contiguous three to six bases including a cytosine-guanine (C-G) sequence linked via a phosphodiester bond, where the spacers other than the spacer(s) containing the CpG site comprise bases other than guanine (G), and 75% or more of the linkages between deoxynucleotides are phosphodiester bonds.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to immunostimulatory oligonucleotides. [Background technology]

[0002] It is known that a sequence consisting of unmethylated cytosine (C) and guanine (G) linked via a phosphodiesterase (CpG) stimulates Toll-like receptor 9 (TLR9) to induce an immune response. Non-Patent Document 1 shows that "2006", a CpG oligodeoxynucleotide having the base sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3', activates human TLR9. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Proceedings of the National Academy of Sciences,2001,vol.98,no.16,p.9237-9242 Summary of the Invention [Problem to be solved by the invention]

[0004] CpG oligodeoxynucleotide "2006" is a single-stranded oligodeoxynucleotide that has an immunostimulatory effect, but has insufficient resistance to nucleases. Therefore, an objective of the present invention is to provide an immunostimulatory single-stranded oligodeoxynucleotide having excellent resistance to nucleases. Another objective of the present invention is to provide a quadruplex structure, an immunostimulatory oligodeoxynucleotide complex, a vaccine adjuvant, a method, a use of the immunostimulatory oligodeoxynucleotide complex, a pharmaceutical composition, a method for treating or preventing cancer, and a method for treating or preventing allergies. [Means for solving the problem]

[0005] As a result of extensive investigations aimed at achieving the above object, the present inventors have found that the above object can be achieved by the following configuration.

[0006] [1] A single-stranded oligodeoxynucleotide consisting of 14 to 100 deoxynucleotides, comprising, as a base sequence, four identical poly-G sites consisting of 2 to 5 consecutive guanines (G), two of the poly-G sites being located at the 5'-terminus and 3'-terminus, respectively, and the remaining poly-G site being located between another poly-G site and a spacer site, the spacer site consisting of one or more bases, at least one of the spacer sites comprising one to five CpG sites consisting of 3 to 6 consecutive bases comprising a cytosine (C)-guanine (G) sequence linked via a phosphodiester, the spacer sites other than the spacer site comprising the CpG site consisting of a base other than guanine (G), and 75% or more of the bonds between the deoxynucleotides being phosphodiester bonds. [2] A single-stranded oligodeoxynucleotide according to [1], in which all of the bonds between the deoxynucleotides are phosphodiester bonds. [3] The single-stranded oligodeoxynucleotide according to [1] or [2], which has 1 to 5 of the CpG sites in the entire single-stranded oligodeoxynucleotide. [4] A single-stranded oligodeoxynucleotide according to any one of [1] to [3], which consists of 22 to 54 deoxynucleotides. [5] The single-stranded oligonucleotide according to any one of [1] to [4], wherein the CpG site is represented by Y1Y2CGY3Y4, wherein Y1 and Y4 are each independently at least one base selected from the group consisting of adenine (A), cytosine (C), guanine (G), and thymine (T), and Y2 and Y3 are each independently adenine (A) or thymine (T). [6] The single-stranded oligodeoxynucleotide according to any one of [1] to [5], wherein the CpG site is GTCGTT. [7] The single-stranded oligodeoxynucleotide according to any one of [1] to [6], wherein the spacer sites other than the spacer site containing the CpG site have the same base sequence. [8] A single-stranded oligodeoxynucleotide according to any one of [1] to [7], having any of the nucleotide sequences of SEQ ID NOs: 1 to 9 and 17 to 22 described below, and sequences in which 1 to 3 bases other than the CG sequence in the spacer portion are deleted, substituted or added in the nucleotide sequence described above. [9] A quadruplex structure consisting of the single-stranded oligodeoxynucleotide according to any one of [1] to [8].

[10] An immunostimulatory oligodeoxynucleotide complex comprising a carrier and the quadruplex structure described in [9] supported on the carrier.

[11] A vaccine adjuvant comprising the immunostimulatory oligodeoxynucleotide complex described in

[10] .

[12] A method for preventing infectious diseases in mammals, including humans, birds, or fish, comprising sequentially or simultaneously administering a detoxified or attenuated antigen derived from a pathogen that causes the infectious disease and the immunostimulatory oligodeoxynucleotide complex described in

[10] , thereby promoting the production of antibodies against the pathogen in the body and acquiring immunity against the infectious disease.

[13] Use of the immunostimulatory oligodeoxynucleotide complex described in

[10] in the manufacture of a vaccine for preventing infectious diseases in mammals, including humans, birds, or fish.

[14] Use of the immunostimulatory oligodeoxynucleotide complex described in

[10] for the prevention of infectious diseases in mammals, including humans, birds, or fish.

[15] Use of the immunostimulatory oligodeoxynucleotide complex described in

[10] in the treatment of cancer or in the manufacture of a preventive vaccine.

[16] Use of the immunostimulatory oligodeoxynucleotide complex described in

[10] for the treatment or prevention of cancer.

[17] A pharmaceutical composition for treating or preventing allergies, comprising the immunostimulatory oligodeoxynucleotide complex described in

[10] .

[18] The pharmaceutical composition described in

[17] , further comprising an allergen or a portion thereof.

[19] Use of the immunostimulatory oligodeoxynucleotide complex described in

[10] in the manufacture of a medicine for treating or preventing allergies.

[20] Use of the immunostimulatory oligodeoxynucleotide complex described in

[10] for the treatment or prevention of allergies.

[21] A method for treating or preventing cancer, comprising sequentially or simultaneously administering a cancer antigen or a portion thereof and the immunostimulatory oligonucleotide complex described in

[10] to induce cytotoxic T cells (CTLs) against the cancer antigen in the body and have them attack cancer cells that present the cancer antigen, thereby treating or preventing cancer.

[22] A method for treating or preventing allergies, comprising administering the immunostimulatory oligonucleotide complex described in

[10] to activate allergen-specific helper 1 T (Th1) cells more than helper 2 T (Th2) cells, thereby treating or preventing allergies. Effect of the Invention

[0007] The present invention provides an immunostimulatory single-stranded oligodeoxynucleotide having excellent resistance to nucleases, and also provides a quadruplex structure, an immunostimulatory oligodeoxynucleotide complex, a vaccine adjuvant, a method, a use of the immunostimulatory oligodeoxynucleotide complex, a pharmaceutical composition, a method for treating or preventing cancer, and a method for treating or preventing allergies. [Brief description of the drawings]

[0008] [Figure 1] This is the result of structural analysis of CpG oligodeoxynucleotide (ODN). Specifically, it is a differential absorption spectrum obtained by subtracting the optical absorption spectrum obtained at 90°C from the optical absorption spectrum obtained at 20°C for each CpG ODN. [Diagram 2] Circular dichroism spectra of each CpG ODN in the presence of 4 mM K+ at 25°C. [Diagram 3] The results are shown in polyacrylamide gel electrophoresis of a 10-bp DNA ladder (lane 1), an oligonucleotide with a linear structure and the same 22-mer length as GD1 (lane 2, sequence number 14), GD1 (lane 3), an oligonucleotide with a linear structure and the same 30-mer length as GD2 (lane 4, sequence number 15), GD2 (lane 5), an oligonucleotide with a linear structure and the same 38-mer length as GD3 (lane 6, sequence number 16), and GD3 (lane 7). [Figure 4] The expression levels of luciferase, a reporter protein, in cell lysates were quantified to investigate the effect of each sample in inducing activation of the transcription factor NF-κB in 293XL-hTLR9 cells, in which the structural gene for human TLR9 has been introduced to constitutively express human TLR9, and in 293XL-null cells, in which human TLR9 is not expressed. Data are mean ± SD (n = 5). * indicates the result of one-way ANOVA followed by Sidak's multiple comparison test for comparison with the Dulbecco's phosphate-buffered saline [D-PBS] control group. **** indicates p < 0.0001, *** indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05. ns indicates p > 0.05, indicating no significant difference. [Figure 5A] This shows the results of polyacrylamide gel electrophoresis (PAGE) analysis of each CpG ODN after incubation with 50% fetal bovine serum (FBS) for 1, 2, 4, and 24 hours. [Figure 5B] FIG. 5B is a graph showing the remaining ODN relative to the amount of ODN at the start of incubation in the test of FIG. 5A. [Figure 6] The uptake of each sample modified with a fluorescent dye into mouse macrophage-like RAW264 cells was measured using the fluorescence intensity of the cells as an index, and the fluorescence intensity derived from each sample bound to the cell surface was calculated and subtracted. -CpG indicates the fluorescence intensity of untreated cells to which no sample modified with a fluorescent dye was added. [Figure 7]Cytokine induction in mouse macrophage-like RAW264 cells stimulated with each sample, and relative mRNA levels of IL-6, IL-12, and IFN-β in mouse macrophage-like RAW264 cells stimulated with 4 μM of each sample for 24 hours. Data are mean ± SD (n=5). * indicates the result of one-way ANOVA followed by Tukey's multiple comparison test for comparison with other groups. ****, p<0.0001, ***, p<0.001, **, p<0.01, *, p<0.05. ns, p>0.05, indicates no significant difference. # indicates the result of one-way ANOVA followed by Dunnett's multiple comparison test for comparison with Dulbecco's phosphate buffered saline [D-PBS] control group. ####, p<0.0001, ###, p<0.001, ##, p<0.01, #, p<0.05. ns indicates p>0.05, which means no significant difference. [Figure 8] Cytokine induction in mouse macrophage-like RAW264 cells stimulated with each sample, and relative mRNA levels of IL-6, IL-12, and IFN-β in mouse macrophage-like RAW264 cells after stimulation with 4 μM of each sample for 24 hours. [Figure 9A] Graph showing the effect of the position and number of CpG sites on the immunostimulatory properties of each sample, including the effect of the CpG site position. Data are mean ± SD (n=5). * indicates the result of one-way ANOVA followed by Tukey's multiple comparison test for comparison with other groups. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.01, * indicates p<0.05. ns indicates p>0.05, which means no significant difference. [Figure 9B] Similar test results to those in FIG. 9A, showing the effect of the distance between the CpG site and the poly-G sequence. [Figure 9C] Similar results to FIG. 9A, showing the effect of the number of CpG sites. [Figure 10A]This figure shows G4 CpG oligodeoxynucleotide (ODN)-induced cytokine production in human peripheral mononuclear cells (PBMCs). The figure shows the effect of incubation time on cytokine induction by G4 CpG ODN in human PBMCs. IL-6 in the supernatant of human PBMCs incubated with 8 μM G4 CpG ODNs for 4, 24, and 48 hours was quantified by ELISA. Data are mean ± SD (n=5). * indicates the results of two-way ANOVA followed by Tukey's multiple comparison test for comparison with other groups. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.01, * indicates p<0.05. [Figure 10B] Figure 1 shows G4 CpG oligodeoxynucleotide (ODN)-induced cytokine production in human peripheral mononuclear cells (PBMCs). Cytokine induction by G4 CpG ODN compared with positive control 2006_PD. IL-6 and IFN-γ in the supernatant from human PBMCs incubated with 8 μM GD3 or 2006_PD for 24 hours were quantified by ELISA. Data are mean ± SD (n=4). * indicates the result of one-way ANOVA followed by Tukey's multiple comparison test for comparison with other groups. * indicates p<0.05. [Figure 11] This figure shows the time course of IL-6 induction by GD3 or 2006_PD in mice. Data are the mean ± SD of 5 mice. * indicates the result of two-way ANOVA followed by Sidak's multiple comparison test. ** indicates p<0.01. ns indicates p>0.05, which means no significant difference. § indicates the lower limit of detection (3.9 pg / mL), and BI indicates before injection. [Figure 12]The results are shown in polyacrylamide gel electrophoresis of a 10-bp DNA ladder (lanes 1 and 12), an oligonucleotide with a linear structure and the same 30-mer length as GD2 (lane 2, sequence number 15), GD2-1L (lane 3, sequence number 17), GD2 (lane 4, sequence number 2), GD2-3L (lane 5, sequence number 19), a nucleotide with a linear structure and the same 38-mer length as GD3 (lane 6, sequence number 16), GD3-1L (lane 7, sequence number 18), GD3 (lane 8, sequence number 3), GD2T-1L (lane 9, sequence number 20), GD2T-2L (lane 10, sequence number 21), and GD2T-3L (lane 11, sequence number 22). [Figure 13] These are the results of polyacrylamide gel electrophoresis of a 10-bp DNA ladder (lane 1), a nucleotide with a linear structure and the same chain length of 38 mer as GD3 (lane 2, column number 16), GD3 (lane 3, sequence number 3), GD3-GC123 (lane 4, sequence number 12), and GD3-GC3 (lane 5, sequence number 9). [Figure 14] The expression level of alkaline phosphatase secreted into the medium was quantified to examine the effect of each sample on inducing activation of the transcription factor NF-κB using RAW-BLUE cells, which are RAW 264.7 macrophages transfected with the secretory embryonic alkaline phosphatase gene induced by the NF-κB transcription factor. Data are the mean ± SD (n = 5). Dulbecco's phosphate-buffered saline [D-PBS] was used as a control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will be described in detail below. The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0010] (single-stranded oligodeoxynucleotide) As used herein, "oligodeoxynucleotide (ODN)" refers to an oligodeoxyribonucleotide, which is composed of multiple structural units (deoxyribonucleotides, hereinafter also referred to simply as "deoxynucleotides") in which phosphate is bound via deoxyribose to a base selected from adenine (A), guanine (G), cytosine (C), and thymine (T).

[0011] The single-stranded ODN of the present invention consists of 14 to 100 deoxynucleotides, preferably 20 to 80 deoxynucleotides, and more preferably 22 to 54 deoxynucleotides.

[0012] In the single-stranded ODN of the present invention, the bonds between deoxynucleotides are phosphodiester bonds in 75% or more of the total number of bonds, preferably 85% or more, more preferably 95% or more, and even more preferably all bonds. In the following explanation of the base sequence of a single-stranded ODN, we will explain the case where all bonds between deoxynucleotides are phosphodiester bonds (represented as a sequence of bases in capital letters); however, as described above, up to 25% of the bonds between oligodeoxynucleotides, preferably up to 15%, and more preferably up to 5%, may include phosphorothioate bonds in which the oxygen atom of the phosphate group of the deoxynucleotide is replaced with a sulfur atom.

[0013] The single-stranded ODN of the present invention contains four identical (sequence) poly-G sites consisting of 2 to 5 consecutive guanines (G). Among them, it is preferable to contain four identical poly-G sites consisting of 3 to 5 consecutive guanines (G), and it is more preferable to contain four identical poly-G sites (GGG) consisting of 3 consecutive guanines.

[0014] Of these, two poly G sites are located at the 5' end and the 3' end, respectively, and the remaining (two) poly G sites are located via spacer sites with other poly G sites. That is, the single-stranded ODN of the present invention is 5'-(poly G site)-(spacer site)-(poly G site)-(spacer site)-(poly G site)-(spacer site)-(poly G site)-3', and has four poly G sites and three spacer sites located between them.

[0015] The spacer site is composed of one or more bases, and generally preferably composed of 5 or less bases, and at least one of the three spacer sites contains 1 to 5 CpG sites each consisting of 3 to 6 consecutive bases including a cytosine (C)-guanine (G) sequence linked via a phosphodiester. Adjacent CpG sites are preferably separated by 1 base or more, and the base is preferably A or T, more preferably T.

[0016] Of the three spacer sites contained in the single-stranded ODN as a whole, it is preferable that the spacer site other than the one containing the CpG site, i.e., the spacer site not containing the CpG site, is composed of a base other than guanine (G) (at least one base selected from the group consisting of A, T, and C).

[0017] The CpG site is preferably represented as Y1Y2CGY3Y4 (5' end side), where Y1 and Y4 are A, C, G, or T, Y2 is A or T, and Y3 is A or T. Among them, Y2 is preferably T and Y3 is preferably T, in that single-stranded ODN has a more excellent effect of the present invention. In particular, when the CpG site is GTCGTT, the effect of the present invention is more excellent.

[0018] The single-stranded ODN of the present invention preferably has 1 to 5 CpG sites as a whole, and it is preferable that one of the spacer sites has 1 to 5 CpG sites, and the remaining two spacer sites do not contain CpG sites.In addition, at this time, it is preferable that the two spacer sites that do not have CpG sites have the same base sequence.

[0019] The single-stranded oligonucleotide of the present invention preferably has any of the following base sequences or sequences in which 1 to 3 bases other than CG in the spacer portion already described above are deleted, substituted or added: In addition, the term "bases other than CG in the spacer region" refers to bases that do not fall into either the poly-G region or "CG" in relation to the entire single-stranded ODN. 5'-GGGTTGGGGTCGTTGGGTTGGG-3' (SEQ ID NO: 1) 5'-GGGTTGGGGTCGTTTTGTCGTTGGGTTGGG-3' (SEQ ID NO: 2) 5'-GGGTTGGGGTCGTTTTGTCGTTTTGTCGTTGGGTTGGG-3' (SEQ ID NO: 3) 5'-GGGTTGGGGTCGTTTTGTm5CGTTGGGTTGGG-3' (SEQ ID NO: 4) 5'-GGGTTGGGGTm5CGTTTTGTCGTTTTGTCGTTGGGTTGGG-3' (SEQ ID NO: 5) 5'-GGGTTGGGGTCGTTTTGTm5CGTTTTGTCGTTGGGTTGGG-3' (SEQ ID NO: 6) 5'-GGGTTGGGGTCGTTTTGTCGTTTTGTm5CGTTGGGTTGGG-3' (SEQ ID NO: 7) 5'-GGGTTGGGGTm5CGTTTTGTCGTTTTGTm5CGTTGGGTTGGG-3' (SEQ ID NO: 8) 5'-GGGTTGGGGTCGTTTTGTCGTTTTGTGCTTGGGTTGGG-3' (SEQ ID NO: 9) 5'-GGGGTCGTTTTGTCGTTGGGTTGGGTTGGG-3' (SEQ ID NO: 17) 5'-GGGGTCGTTTTGTCGTTTTGTCGTTGGGTTGGGTTGGG-3' (SEQ ID NO: 18) 5'-GGGTTGGGTTGGGGTCGTTTTGTCGTTGGG-3' (SEQ ID NO: 19) 5'-GGGGTCGTTTTGTCGTTGGGTGGGTGGG-3' (SEQ ID NO: 20) 5'-GGGTGGGGTCGTTTTGTCGTTGGGTGGG-3' (SEQ ID NO: 21) 5'-GGGTGGGTGGGGTCGTTTTGTCGTTGGG-3' (SEQ ID NO: 22)

[0020] In the above sequence, m5C indicates methylated cytosine (5-methylcytosine). In the above sequence, all bonds between deoxynucleotides are phosphodiester bonds, but as described above, some may be phosphorothioate bonds or the like. In this specification, capitalized bases mean that the bonds between deoxynucleotides are phosphodiester bonds. "m5C" is treated as a single capital letter.

[0021] Single-stranded ODN can be synthesized using a nucleic acid synthesizer. Alternatively, a method of amplifying a region containing CpG by PCR using bacterial or viral genome ODN as a template can be used. Alternatively, circular plasmid ODN can be amplified in a host cell, and the collected circular plasmid can be cleaved with a restriction enzyme to obtain the ODN.

[0022] The single-stranded ODN of the present invention has four pairs of poly-G sites. Therefore, a quadruplex structure (guanine quadruplex structure) consisting of a single strand is easily induced. The quadruplex structure consisting of the single-stranded ODN of the present invention is preferably composed of only single-stranded ODN. The quadruplex structure consisting of single-stranded ODN (hereinafter also referred to as "G4 CpG ODN") is known to have the topology of "parallel", "antiparallel" and "hybrid". The quadruplex structure of the present invention may be in any form, but the "hybrid" type is preferred.

[0023] The quadruplex structure of the present invention has excellent resistance to nucleases and, surprisingly, due to its compact structure, has excellent absorbability into cells. As a result, it has a superior immunostimulatory effect compared to linear single-stranded CpG ODN.

[0024] The method of inducing a quadruplex structure from a single-stranded ODN is not particularly limited, and known methods such as those described in the Examples below can be used. Specifically, the stability of the quadruplex structure is improved by incorporating a monovalent metal ion (cation) into the inside of the quadruplex structure, so a method of heating in the presence of an ion source compound can be used. Examples of monovalent metal ions include potassium ions and sodium ions.

[0025] <Immune stimulating ODN complex> The second aspect of the present invention is an immunostimulatory ODN complex in which the quadruplex structure composed of the single-stranded ODN prepared as described above is complexed with a physiologically acceptable carrier. By complexing, the effect of the present invention can be more excellent. The term "physiologically acceptable carrier" refers to a substance that does not cause damage to cells, tissues, or organs in the body that would impede the objectives of the present invention. Examples of such carriers include polymer compounds, emulsions, liposomes, inorganic compound particles, metal particles, metal oxide particles, carbon-based particles, and modified products thereof, and are preferably cationic.

[0026] Examples of polymer compounds include cationic polymers such as polyethyleneimine, chitosan, polylysine, LL-37, and Tat. These cationic polymers electrostatically bind to the quadruplex structure of the present invention. There is also a method of encapsulating the quadruplex structure in poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer. In addition, polymers such as dendrimers can also be used as carriers.

[0027] Examples of emulsions include water / oil emulsions and water / oil / water emulsions, and a method of encapsulating a quadruplex structure in the aqueous phase is possible. Examples of liposomes include a method of encapsulating a quadruplex structure in a liposome composed of a lipid bilayer membrane, and a method of electrostatically binding a quadruplex structure to a cationic liposome, for example, one containing a long-chain alkyl group and an amino group or an ammonium group.

[0028] Examples of inorganic compound particles include calcium phosphate particles, apatite hydroxide particles, apatite carbonate particles, and silica nanoparticles. Examples of metal particles include gold particles, silver particles, platinum particles, and silicon nanoparticles. Examples of metal oxide particles include zinc oxide particles, titanium dioxide particles, alumina particles, and zirconia particles. Examples of carbon-based particles include fullerenes, carbon nanotubes, and carbon nanohorns.

[0029] The size of these carrier particles is not particularly limited, but the average length or longest diameter (D50) is preferably 10 nm to 1 μm, more preferably 100 to 800 nm. The particle shape is not particularly limited, and may be spherical, flake-like, cylindrical, etc.

[0030] Of the above carriers, cationic liposomes and inorganic compound particles are preferred. Cationic liposomes containing a long-chain alkyl group and an amino group or an ammonium group, such as Lipofectamine (trademark), DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate), DMTAP (dimyristoyltrimethylammonium propane), DOAB (dimethyldioctadecylammonium (bromide salt)), DODAP (1,2-dioleoyl-3-dimethylammonium-propane), DC-CHOL (3b-[N-(N',N'-dimethylaminoethane)-caramoyl]cholesterol hycrochloride), DOSPA (N-[2-[(1,5,10,14-Tetraazatetradecane-1-yl)carbonylamino]ethyl]-N,N-dimethyl-2,3-bis(oleoyloxy)-1-propanaminium), or a mixture with uncharged DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamin) (for example, DOSPA / DOPE (3:1 wt / wt)), and calcium phosphate and DEAE dextran are preferred as inorganic compounds. Alternatively, a mixture of these is preferred.

[0031] The method of complexing the quadruplex structure of the present invention with these carriers is not particularly limited. The quadruplex structure can be electrostatically bound to a carrier having a positively charged surface by mixing it in an appropriate buffer. The quadruplex structure is adsorbed to calcium phosphate particles, hydroxyapatite particles, carbonate apatite particles, fullerene, carbon nanotubes, and carbon nanohorns. In addition, as a method of supporting a carrier having a non-positively charged surface, there is a method of electrostatically binding the quadruplex structure by modifying the surface of the carrier particle with a positively charged substance such as polyethyleneimine, chitosan, polylysine, etc., or a method of introducing a maleimide group to the surface of the carrier particle and, on the other hand, introducing a thiol group to the end of the quadruplex structure to form a covalent bond.

[0032] The mass ratio of the quadruplex structure to the carrier is preferably adjusted appropriately depending on the base chain length of the single-stranded oligonucleotide, the properties of the carrier, the antigen used together, etc. Typically, the mass ratio of the quadruplex structure to the carrier is preferably 0.05:1 to 10:1.

[0033] The effect of the immunostimulatory ODN complex can be determined by quantifying the expression level of type I IFN or inflammatory cytokine genes or the secretion level of type I IFN or inflammatory cytokines after administering the complex to cells having TLR9, such as human and mouse plasmacytoid dendritic cells, mouse macrophages, and mouse stationary dendritic cells. For example, the gene expression level can be quantified by real-time quantitative PCR, and the secretion level can be quantified by ELISA.

[0034] The immunostimulatory ODN complex is expected to exert an adjuvant effect by being given together with an antigen or an allergen. The complex can be administered by mixing with a free antigen or allergen. In addition, there is a method in which the antigen or allergen is administered by conjugating it to the same carrier as the complex, or a method in which the antigen or allergen is first bound to a single-stranded ODN and then conjugated to a carrier and administered. In addition, there is a method in which the antigen or allergen is administered by conjugating it to a carrier other than the complex and mixing it with the complex.

[0035] The method for complexing an antigen or allergen with a carrier is not particularly limited. For example, a method in which an antigen or allergen is encapsulated in the hollow space of a cationic liposome and then a quadruplex structure is electrostatically bound to the surface of the cationic liposome, or a method in which a quadruplex structure and an antigen or allergen are simultaneously adsorbed onto the surface of calcium phosphate particles, apatite hydroxide particles, or apatite carbonate particles, may be used.

[0036] In addition, by mixing the quadruplex structure and the antigen or allergen in the raw material solution when preparing calcium phosphate particles, hydroxyapatite particles, or carbonate apatite particles, the quadruplex structure and the antigen or allergen can be simultaneously encapsulated in these particles. When the antigen or allergen is bound to the quadruplex structure in advance and then supported on the carrier, there is a method in which the terminal of the quadruplex structure is modified with a thiol group, while a maleimide group is introduced to the amino group of the antigen or allergen, and the thiol group and the maleimide group are covalently bonded to each other.

[0037] Examples of antigens include SARS-CoV-2 antigens, hand, foot and mouth disease virus antigens, dengue fever virus antigens, and West Nile fever virus antigens. Examples of allergens include cedar pollen allergens, ragweed allergens, rice allergens, and mite allergens. Cancer cell antigens are also included.

[0038] A vaccine is a medicine used to prevent infectious diseases. By administering a detoxified or weakened antigen, the body is encouraged to produce antibodies against pathogens, and immunity against infectious diseases is acquired. Although not limited to these, live vaccines using weakened microorganisms or viruses include BCG, live oral polio (OPV) vaccine, smallpox vaccine, measles vaccine, rubella vaccine, measles-rubella combined vaccine (MR vaccine), mumps vaccine, chickenpox vaccine, yellow fever vaccine, rotavirus vaccine, and shingles vaccine. Inactivated vaccines, which include viruses, bacteria, and rickettsia that have been killed by chemical treatment or that are cultured with only the antigenic parts, include influenza virus vaccines, pneumococcal vaccines, rabies vaccines, cholera vaccines, diphtheria-tetanus (DT) vaccines, diphtheria-pertussis-tetanus (DPT) vaccines, diphtheria-pertussis-tetanus (DPT-IPV) vaccines, Japanese encephalitis vaccines, and pertussis vaccines.

[0039] Cancer vaccines refer to vaccines used for the purpose of treating or preventing cancer. The antigen (cancer antigen) used is a full-length or partial antigen protein (peptide) that is not expressed at all in normal cells or is expressed only in small amounts, but is overexpressed in cancer cells. Examples include, but are not limited to, MAGE in malignant melanoma, HER2 / neu in breast cancer, CEA in colon cancer, WT1 in various leukemias and various cancers, and NY-ESO-1 in malignant melanoma, esophageal cancer, gastric cancer, ovarian cancer, etc. These cancer antigen proteins (or their decomposition peptides) are recognized by cytotoxic T cells (CTL) in the body and attack cancer cells (cellular immunity). Cancer vaccine therapy is a method of treating cancer by artificially administering cancer antigens (peptides) and inducing specific CTLs (differentiation and proliferation). Cancer vaccines also include vaccines aimed at preventing infection with cancer-causing viruses. Vaccines against hepatitis B virus (which can lead to liver cirrhosis and liver cancer) and human papillomavirus (which can cause cervical cancer) are currently in practical use.

[0040] In patients with hay fever or house dust allergy, the T helper 2 (Th2) pathway specific to each allergen predominates over the T helper 1 (Th1) pathway. In other words, one of the therapeutic strategies is to activate the allergen-specific Th1 pathway and make it dominant over the Th2 pathway. In mice, the IgG2a / IgG1 ratio is an indicator of the Th1 / Th2 ratio, while in humans, the IgG4 / IgG1 ratio may be an indicator of the Th1 / Th2 ratio.

[0041] Subjects to be treated in the present application include mammals including humans (primates such as monkeys; companion animals such as dogs and cats; livestock such as horses, pigs, cows, goats, and sheep; and laboratory animals such as rats and mice), birds (wild birds or poultry such as chickens and turkeys), or fish (farmed species: for example, freshwater fish such as sweetfish and salmonids, and saltwater fish such as yellowtail and amberjack), and are not particularly limited as long as the species expresses TLR9. EXAMPLES

[0042] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0043] (Intramolecular G-quadruplex formation) Single-stranded CpG ODNs having the base sequences of SEQ ID NOs: 1 to 9 and 17 to 22 in Table 1 were synthesized and subjected to heat treatment to form a guanine quadruplex structure (G4 CpG ODN). Specifically, the single-stranded CpG ODNs were dissolved in a buffer solution (Dulbecco's phosphate-buffered saline: hereinafter also referred to as "D-PBS") containing 2.68 mM KCl, 137 mM NaCl, 1.47 mM KH2PO4, and 8.10 mM Na2HPO4, and incubated at 95°C for 5 minutes, and then the temperature was lowered to 30°C over 30 minutes. For comparison, single-stranded ODNs of SEQ ID NOs: 10 to 16 in Table 1 were also prepared.

[0044] [Table 1]

[0045] (Absorption spectrum analysis) Single-stranded CpG ODN that had formed a G-quadruplex structure by heat treatment was prepared at 2 μM using D-PBS. The absorption spectrum was measured at 220-320 nm using a Jasco V-670 spectrophotometer. The measurement conditions were a scan speed of 400 nm / min, a data pitch of 1 nm, and a band width of 2.0 nm, and UV / Vis (ultraviolet / visible) measurement. Measurements were performed at 20°C and 90°C, and the absorption spectrum at 90°C was subtracted from the absorption spectrum at 20°C to obtain a difference spectrum. Figure 1 shows the difference absorption spectra of GD1, GD2, and GD3.

[0046] As shown in FIG. 1, GD1, GD2, and GD3 all showed a negative peak at 295 nm, indicating that they formed a G-quadruplex structure.

[0047] (Circular dichroism spectrum analysis) Single-stranded CpG ODN that had formed a G-quadruplex structure by heat treatment was prepared at 2uM using D-PBS. Circular dichroism spectra from 220 to 320nm were measured at 25℃ using a circular dichroism spectropolarimeter (Jasco J-725 CD spectropolarimeter). The measurement conditions were: scan rate: 50nm / min, response time: 2.0s, band width: 1.0nm, resolution: 0.2nm, and sensitivity: 100mdeg.

[0048] FIG. 2 shows the circular dichroism spectra of GD1, GD2, and GD3. As shown in Figure 2, GD1 and GD2 showed positive peaks at 260 nm and 290 nm, and GD3 had a broad peak spanning 260 nm and 290 nm, indicating that it formed a hybrid G-quadruplex structure.

[0049] (Structural analysis by electrophoresis) Electrophoresis was performed on 12% polyacrylamide gels (PAGE; 1-mm thick; TEFCO, Tokyo, Japan) in 0.5× Tris-borate-EDTA (TBE) buffer (0.089 M, pH 8.3-8.5; TakaraBio, Kusatsu, Japan) containing 4 mM KCl at a constant voltage of 180 V.

[0050] Figure 3 shows the results of PAGE electrophoresis. Lane 1 is a 10-bp DNA ladder, lane 2 is an oligonucleotide with a linear structure and a chain length of 22 mers, the same as GD1, lane 3 is GD1, lane 4 is an oligonucleotide with a linear structure and a chain length of 30 mers, the same as GD2, lane 5 is GD2, lane 6 is an oligonucleotide with a linear structure and a chain length of 38 mers, the same as GD3, and lane 7 is GD3.

[0051] The results in Figure 3 indicate that GD1, GD2, and GD3 are located at low molecular weight positions compared to linear oligonucleotides of the same chain length, indicating that GD1, GD2, and GD3 form a compactly folded structure (G-quadruplex structure).

[0052] (Evaluation of immune activation using HEK293 cells) The structural gene for human TLR9 was inserted into HEK293 cells, a cell line established by transforming human fetal kidney cells with the E1 gene of adenovirus, to constitutively express human TLR9. In 293XL-hTLR9 cells, immune activation was evaluated by investigating the effect of inducing activation of the transcription factor NF-κB.

[0053] 293XL-hTLR9 cells were cultured at 1.3 × 10 6 The cells were seeded in Dulbecco's modified Eagle medium (DMEM) at a concentration of 1000 cells / ml. The reporter assay plasmid pNiFty-luc (Invivogen) for investigating the activation of the transcription factor NF-κB and the plasmid pGL4.74 (Promega) for standardization of gene transfer were introduced using the transfection reagent LyoVec (Invivogen). After 20 hours, GD1, GD2, GD3, and 2006_PD were added to a final concentration of 0.5 μM. 24 hours after the addition of each sample, the cells were harvested and disrupted, and the expression level of luciferase, a reporter protein, in the cell lysate was quantified.

[0054] Figure 4 shows the results of quantifying the expression level of luciferase in the cell lysate obtained. The CpG oligonucleotides GD1, GD2, and GD3 that formed a G-quadruplex structure induced the activation of NF-κB in 293XL-hTLR9 cells. Furthermore, NF-κB activation was not induced in 293XL-null cells, which do not express human TLR9. This indicates that the CpG oligonucleotides GD1, GD2, and GD3 that formed a G-quadruplex structure induce the activation of the transcription factor NF-κB via human TLR9. Furthermore, it was shown that the activation of the transcription factor NF-κB was higher than that of the linear CpG ODN "2006_PD."

[0055] (Stability in serum) The stability of GD1, GD2, and GD3 that formed a G-quadruplex structure in fetal bovine serum containing nuclease was quantified using electrophoretic bands as an index. Fetal bovine serum was prepared to a final concentration of 50%, and GD1, GD2, and GD3 that formed a G-quadruplex structure in it, as well as linear CpG ODN2006_PD for comparison, were incubated for 1 hour, 2 hours, 4 hours, and 24 hours. Then, electrophoresis was performed, and the intensity of the bands derived from each oligonucleotide was quantified to determine the amount of undegraded oligonucleotide, and the remaining amount was calculated by taking the sample before serum treatment as 100%. Figures 5A and 5B show the results.

[0056] Figures 5A and 5B show that the linear CpG ODN "2006_PD" was almost completely degraded after 1 hour in fetal bovine serum, whereas 81%, 50%, and 32% of the CpG oligonucleotides GD1, GD2, and GD3 that formed a G-quadruplex structure remained undegraded, respectively. Even after 4 hours of treatment in fetal bovine serum, GD1, GD2, and GD3 were observed to remain undegraded. This indicates that GD1, GD2, and GD3 that formed a G-quadruplex structure have resistance to nucleases compared to linear CpG ODN.

[0057] (Cellular uptake) The uptake of GD2 and GD3, which had G-quadruplex structures formed by modifying the 5'-end with the fluorescent dye Cy5, into mouse macrophage-like RAW264 cells was measured using the fluorescence intensity of the cells as an indicator. GD1 and GD2, which had G-quadruplex structures formed by modifying the 5'-end with Cy5, were added to Opti MEM at a final concentration of 5 μM.

[0058] This solution was added to a 96-well plate with RAW264 cells attached, and incubated at 37°C for 2 hours. After that, the solution was removed and the cells were washed twice with PBS. Next, the cells were detached using trypsin-EDTA (ethylenediaminetetraacetic acid), collected by centrifugation, and suspended in PBS. The amount of Cy5-modified CpG ODN taken up by the cells was analyzed using the fluorescence derived from Cy5 as an index using a Sony Spectral Cell Analyzer SP6800. In addition, samples were prepared by incubating 5'-end Cy5-modified GD1 and GD2 with RAW264 cells at 4°C for 2 hours, and the fluorescence intensity of the CpG oligonucleotides GD1 and GD2 bound to the cell surface was measured. The amount of 5'-end Cy5-modified CpG ODN taken up into the cells was quantified by subtracting the fluorescence intensity. Figure 6 shows the results.

[0059] Figure 6 shows that the 5'-end Cy5-modified CpG oligonucleotides GD1 and GD2 were taken up by RAW cells. In addition, it was shown that the CpG ODN that formed a G-quadruplex structure was taken up more by cells than the linear CpG ODN "2006_PD."

[0060] (Evaluation of immune activation using mouse macrophage-like RAW264 cells) Using RAW264 cells expressing Toll-like receptor 9, we investigated the induction of interleukin 6 (IL-6), IL-12, and interferon (IFN)-β induced by GD1, GD2, and GD3 that formed G-quadruplex structures. RAW264 cells were cultured at 5.3 × 10 5 The cells were seeded in Eagle's minimum essential medium (MEM) at a density of 10 ...

[0061] FIG. 7 shows that GD2 and GD3, which formed a G-quadruplex structure, induced significantly high levels of IL-6, IL-12, and IFN-β.

[0062] In order to show that the effect of CpG ODN that formed a G-quadruplex structure is due to the CpG site introduced into the loop region of the G-quadruplex structure, an experiment similar to that shown in Figure 7 was performed using GD2-Met and GD3-Met, which are ODNs that formed a G-quadruplex structure in which the cytosine (C) at the CpG site was methylated and replaced with 5-methylcytosine. Figure 8 shows the results.

[0063] As shown in Figure 8, methylation of cytosines in the CpG sites of GD2 and GD3, which had formed a G-quadruplex structure, did not induce IL-6, IL-12, or IFN-β genes. It has been reported that Toll-like receptor 9 binds to unmethylated CpG sites and changes the structure to transmit signals. This indicates that the G-quadruplex structure itself does not have the function of activating the immune system, but that the CpG sites introduced into the G-quadruplex structure have the function of activating the immune system.

[0064] To examine in more detail the effect of multiple CpG sites in GD2 and GD3 that formed a G-quadruplex structure, we investigated the induction of IL-6, IL-12, and IFN-β genes by oligonucleotides that formed a G-quadruplex structure in which one or two cytosines were replaced with 5-methylcytosine. Figures 9A, 9B, and 9C show the results. Figures 9A to 9C show that IL-6, IL-12, and IFN-β are induced if there is even one CpG site.

[0065] (Evaluation of immune activation using human peripheral blood mononuclear cells) Using human peripheral blood mononuclear cells purchased from Cellular Technology Limited (Ohio, USA), we investigated the induction of interleukin-6 (IL-6), IL-12, and IFN-β induced by GD1, GD2, and GD3 that had formed G-quadruplex structures. 2.6 × 10 human peripheral blood mononuclear cells 7 The cells were seeded in Roswell Park Memorial Institute medium (RPMI 1640) at a concentration of 1000 cells / ml. GD1, GD2, and GD3, which formed G-quadruplex structures, were added to a final concentration of 4 μM.

[0066] After 6, 24, and 48 hours, the induction levels of IL-6, IFN-γ, and IFN-α were measured using IL-6 Ready-Set-Go ELISA kits (Thermo Fisher Science), IFN-γ Ready-Set-Go ELISA kits (Thermo Fisher Science), and Human IFN-α Module Set enzyme-linked immunosorbent assay (ELISA) (Thermo Fisher Science). The results are shown in Figures 10A and 10B.

[0067] Figures 10A and 10B show that the addition of GD2 and GD3, which had formed a G-quadruplex structure, induced the expression of IL-6 in human peripheral blood mononuclear cells 24 and 48 hours later. The amount of IL-6 induction was highest 24 hours after the addition of the CpG oligonucleotide. It was also shown that GD3, which had formed a G-quadruplex structure, induced IL-6 and IFN-γ at a significantly higher level than the linear CpG ODN "2006_PD". GD3 has the same number of CpG sites in the loop region as 2006_PD. This shows that the immune activation function is significantly improved by using the G-quadruplex structure.

[0068] (Evaluation of immune activation in vivo using mice) The amount of IL-6 induced in the blood was evaluated by administering GD3 with G-quadruplex structure to mice (C57BL / 6J). 10 nmol of GD3 with G-quadruplex structure or linear CpG ODN "2006_PD" was administered to the abdominal cavity of 7-week-old female mice (C57BL / 6). Blood was collected from the vein located behind the jaw bone using an animal lancet every 2, 4, and 24 hours after administration. The collected blood was treated with EDTA and centrifuged to prepare plasma. The amount of IL-6 in the plasma was measured using IL-6 Ready-Set-Go ELISA kits (Thermo Fisher Science). The results are shown in Figure 11.

[0069] Figure 11 shows that IL-6 was induced 2 and 4 hours after administration. The amount of IL-6 induction was highest 4 hours after addition of CpG oligonucleotide. It was also shown that GD3, which formed a G-quadruplex structure, induced IL-6 significantly more than the linear CpG ODN "2006_PD."

[0070] (Structural analysis by electrophoresis) Electrophoresis was performed on 10-20% polyacrylamide gels (PAGE; 1-mm thick; ATTO, Tokyo, Japan) in 25 mM Tris-192 mM Glysin buffer containing 4 mM KCl at a constant current of 21 mA.

[0071] Figure 12 shows the results of PAGE electrophoresis. Lane 1 (and lane 12) is a 10-bp DNA ladder, lane 2 is an oligonucleotide (SEQ ID NO: 15) with a chain length of 30 mer and linear structure, which is the same as GD2 (SEQ ID NO: 2), GD2-1L (SEQ ID NO: 17), and GD2-3L (SEQ ID NO: 19), lane 3 is GD2-1L (SEQ ID NO: 17), lane 4 is GD2 (SEQ ID NO: 2), and lane 5 is GD2-3L (SEQ ID NO: 19). Lane 6 is a nucleotide (SEQ ID NO: 16) with a chain length of 38 mer and linear structure, which is the same as GD3 (SEQ ID NO: 3) and GD3-1L (SEQ ID NO: 18), lane 7 is GD3-1L (SEQ ID NO: 18), and lane 8 is GD3 (SEQ ID NO: 3). Lane 9 is GD2T-1L (sequence number 20) with a chain length of 28 mer, lane 10 is GD2T-2L (sequence number 21) with a chain length of 28 mer, and lane 11 is GD2T-3L (sequence number 22) with a chain length of 28 mer.

[0072] The results in Figure 12 show that GD2-1L, GD2, GD2-3L, GD3-1L, and GD3 are located at lower molecular weights than linear oligonucleotides of the same length. Also, compared to the 10-bp DNA ladder, GD2T-1L, GD2T-2L, and GD2T-3L, which are 28-mer in length, are located at lower molecular weights. This indicates that GD2-1L, GD2, GD2-3L, GD3-1L, GD3, GD2T-1L, GD2T-2L, and GD2T-3L form compactly folded structures (G-quadruplex structures).

[0073] (Structural analysis by electrophoresis) Electrophoresis was performed on 10-20% polyacrylamide gels (PAGE; 1-mm thick; ATTO, Tokyo, Japan) in 25 mM Tris-192 mM Glysin buffer containing 4 mM KCl at a constant current of 21 mA.

[0074] 13 shows the results of PAGE electrophoresis. Lane 1 is a 10-bp DNA ladder, lane 2 is GD3 (SEQ ID NO: 3), GD3-GC123 (SEQ ID NO: 12), and an oligonucleotide (SEQ ID NO: 16) with a linear structure and a chain length of 38 mer, which is the same as GD3-GC3 (SEQ ID NO: 9), lane 3 is GD3 (SEQ ID NO: 3), lane 4 is GD3-GC123 (SEQ ID NO: 12), and lane 5 is GD3-GC3 (SEQ ID NO: 9).

[0075] The results in Figure 13 indicate that GD3, GD3-GC123, and GD3-GC3 are located at lower molecular weights than linear oligonucleotides of the same length, indicating that GD3, GD3-GC123, and GD3-GC3 form compactly folded structures (G-quadruplex structures).

[0076] (Evaluation of immune activation using RAW-BLUE cells) Immune activation (immunostimulatory activity) was evaluated by examining the effect of inducing activation of the transcription factor NF-κB in RAW-BLUE cells (Invivogen), which are RAW 264.7 macrophages incorporating the secretory embryonic alkaline phosphatase gene induced by the NF-κB transcription factor.

[0077] RAW-BLUE cells, 1.0 x 10 5 The cells were seeded in Dulbecco's modified Eagle medium (DMEM) so that each cell was present in one well of a 96-well plate. Immediately after seeding, GD2-1L, GD2, GD2-3L, GD3-1L, GD3, GD2T-1L, GD2T-2L, and GD2T-3L were added to the wells at a final concentration of 2.0 μM. 24 hours after the addition of each sample, the culture medium was collected. The alkaline phosphatase in the culture medium was quantified using QUANTI-Blue solution (Invivogen), a substrate reagent for alkaline phosphatase.

[0078] Figure 14 shows the results of quantifying the expression level of alkaline phosphatase in the culture medium obtained. The CpG oligonucleotides GD2-1L, GD2, GD2-3L, GD3-1L, GD3, GD2T-1L, GD2T-2L, and GD2T-3L that formed a G-quadruplex structure induced NF-κB activation in RAW-BLUE cells. This indicates that the immune activation function is significantly improved by using the CpG oligonucleotides that formed a G-quadruplex structure. [Industrial Applicability]

[0079] The single-stranded ODN of the present invention can form a G-quadruplex structure in the monomer. This indicates that the higher-order structure can be controlled. The guidelines of the US Food and Drug Administration (FDA) published in 2014 recommend that manufacturers reduce the amount of aggregates as much as possible from the perspective of risk management. They also point out that it is very important to monitor changes in aggregates during storage. Since aggregates are thought to occur and grow over time, even if they are sufficiently removed at the time of shipping, there is a risk that aggregates will increase again during the subsequent transportation, storage, preparation, and administration processes. In this respect, the single-stranded ODN of the present invention, which can control the higher-order structure, is superior in that various higher-order structures are not mixed, compared to conventional methods that improve stability by combining multiple oligonucleotides to form DNA three-dimensional structures such as dendrimers.

[0080] In addition, in one form of the single-stranded ODN of the present invention, all of the bonds between deoxynucleotides are phosphodiester bonds, and side effects that are a concern in oligonucleotides having conventional phosphorothioate backbones are less likely to occur.

[0081] Furthermore, the quadruplex structure of the single-stranded ODN of the present invention has excellent resistance to nucleases and, due to its compact structure, has excellent cell absorption. As a result, it has a superior immunostimulatory effect compared to conventional linear single-stranded ODN.

[0082] The immunostimulatory oligodeoxynucleotide complex of the present invention, which has all of the above-mentioned characteristics, is useful as a therapeutic drug for allergies and the like, and as a vaccine adjuvant, etc.

Claims

1. A quadruplex structure composed only of single-stranded oligodeoxynucleotides each consisting of 22 to 100 deoxynucleotides, The base sequence of the single-stranded oligodeoxynucleotide is four poly-G sites (GGG) consisting of three consecutive guanines (G); and three spacer portions each consisting of one or more bases; Among the four poly-G sites (GGG), two are located at the 5' end and the 3' end, respectively, and the remaining two poly-G sites are located with the spacer site interposed between them and the other poly-G sites; any one of the three spacer sites contains 1 to 5 CpG sites, and the remaining two spacer sites do not contain the CpG site and are composed of a base other than guanine (G); the base sequence of the CpG site is GTCGTT, At least 75% of the bonds between the deoxynucleotides are phosphodiester bonds; The base sequence of the single-stranded oligodeoxynucleotide is A quadruplex structure composed only of single-stranded oligodeoxynucleotides, having either (i) the following base sequence, or (ii) the following base sequence in which 1 to 3 bases other than the CpG site are deleted, substituted or added in the spacer site, and the spacer site after the deletion, substitution or addition is a sequence consisting of one or two or more bases. 5'-GGGTTGGGGTCGTTGGGTTGGG-3' (SEQ ID NO: 1) 5'-GGGTTGGGGTCGTTTTGTCGTTGGGTTGGG-3' (SEQ ID NO: 2) 5'-GGGTTGGGGTCGTTTTGTCGTTTTGTCGTTGGGTTGGG-3' (SEQ ID NO: 3) 5'-GGGTTGGGGTCGTTTTGTm5CGTTGGGTTGGG-3' (SEQ ID NO: 4) 5'-GGGTTGGGGTm5CGTTTTGTCGTTTTGTCGTTGGGTTGGG-3' (SEQ ID NO:5) 5'-GGGTTGGGGTCGTTTTGTm5CGTTTTGTCGTTGGGTTGGG-3' (SEQ ID NO: 6) 5'-GGGTTGGGGTCGTTTTGTCGTTTTGTm5CGTTGGGTTGGG-3' (SEQ ID NO: 7) 5'-GGGTTGGGGTm5CGTTTTGTCGTTTTGTm5CGTTGGGTTGGG-3' (SEQ ID NO: 8) 5'-GGGTTGGGGTCGTTTTGTCGTTTTGTGCTTGGGTTGGG-3' (SEQ ID NO: 9) 5'-GGGGTCGTTTTGTCGTTGGGTTGGGTTGGG-3' (SEQ ID NO: 17) 5'-GGGGTCGTTTTGTCGTTTTGTCGTTGGGTTGGGTTGGG-3' (SEQ ID NO: 18) 5'-GGGTTGGGTTGGGGTCGTTTTGTCGTTGGG-3' (SEQ ID NO: 19) 5'-GGGGTCGTTTTGTCGTTGGGTGGGTGGG-3' (SEQ ID NO: 20) 5'-GGGTGGGGTCGTTTTTGTCGTTGGTGGG-3' (SEQ ID NO:21) 5'-GGGTGGGTGGGGTCGTTTTTGTCGTTGGG-3' (SEQ ID NO:22) (In the above sequence, m5C indicates a methylated cytosine.)

2. The quadruplex structure composed only of single-stranded oligodeoxynucleotides according to claim 1, wherein all of the bonds between the deoxynucleotides are phosphodiester bonds.

3. A quadruplex structure composed only of the single-stranded oligodeoxynucleotide described in claim 1 or 2, wherein the single-stranded oligodeoxynucleotide consists of 22 to 54 deoxynucleotides.

4. A quadruplex structure composed only of the single-stranded oligodeoxynucleotide according to any one of claims 1 to 3, wherein the remaining two spacer sites have the same base sequence.

5. A carrier and the quadruplex structure according to any one of claims 1 to 4 supported on the carrier, The immunostimulatory oligodeoxynucleotide complex, wherein the carrier is at least one selected from the group consisting of polymer compounds, emulsions, liposomes, inorganic compound particles, metal particles, metal oxide particles, carbon-based particles, and modified products thereof.

6. A vaccine adjuvant comprising the immunostimulatory oligodeoxynucleotide complex of claim 5.

7. A method for preventing infectious diseases in mammals other than humans, birds, or fish, comprising sequentially or simultaneously administering a detoxified or attenuated antigen derived from a pathogen that causes the infectious disease and the immunostimulatory oligodeoxynucleotide complex described in claim 5, thereby promoting the production of antibodies against the pathogen in the body and acquiring immunity against the infectious disease.

8. Use of the immunostimulatory oligodeoxynucleotide complex of claim 5 in the manufacture of a vaccine for preventing infectious diseases in mammals, including humans, birds, or fish.

9. Use of the immunostimulatory oligodeoxynucleotide complex according to claim 5 for the prevention of infectious diseases in mammals other than humans, birds or fish.

10. Use of the immunostimulatory oligodeoxynucleotide complex of claim 5 in the production of a cancer therapeutic vaccine or a cancer preventive vaccine.

11. A pharmaceutical composition for treating cancer or for preventing cancer, comprising the immunostimulatory oligodeoxynucleotide complex of claim 5.

12. A pharmaceutical composition for treating allergies or for preventing allergies, comprising the immunostimulatory oligodeoxynucleotide complex of claim 5.

13. The pharmaceutical composition according to claim 12, further comprising an allergen or a part thereof.

14. 6. Use of the immunostimulatory oligodeoxynucleotide complex of claim 5 in the manufacture of a medicament for treating allergies or in the manufacture of a medicament for preventing allergies.

15. Further comprising a cancer antigen or a portion thereof, The pharmaceutical composition for cancer treatment or the pharmaceutical composition for cancer prevention according to claim 11, which, when administered, induces cytotoxic T cells (CTL) against a cancer antigen in the body and attacks cancer cells that present the cancer antigen, thereby treating or preventing cancer.

16. A pharmaceutical composition for treating allergies or a pharmaceutical composition for preventing allergies as described in claim 12, which, when administered, activates allergen-specific helper 1 T (Th1) cells more than helper 2 T (Th2) cells, thereby treating or preventing allergies.

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