TW201639583A - CpG-spacer-oligonucleotide-containing complex having immunostimulating activity and use thereof - Google Patents

Abstract

The present invention provides an oligodeoxynucleotide containing humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located on the 3' side of the humanized K-type CpG oligodeoxynucleotide with a spacer interposed therebetween. The present invention also provides a complex containing this oligodeoxynucleotide and [beta]-1,3-glucan.

Description

CpG-spacer-oligonucleotide complex having immunostimulating activity and use thereof

The present invention relates to an oligonucleotide-containing complex having immunostimulating activity, a use thereof, and an oligonucleotide for forming the complex. In particular, the present invention relates to a complex comprising CpG oligodeoxynucleotide (ODN) and β-glucan (glucan) having immunostimulating activity, and pharmaceutical use thereof, and for forming the complex ODN.

CpG oligodeoxynucleotide (CpG ODN) is a CpG motif containing an immunostimulating activity (immune-active) (the rate of occurrence of a CpG site is higher in the genome than other) (about 20 base pairs) single-stranded DNA fragment, a powerful agonist of Toll-like receptor 9 (TLR9), dendritic cells (DCs) And B cells are activated, and type I interferons (IFNs) and cytokine are produced (Non-Patent Documents 1 and 2) as a Th1 type containing a cytotoxic T lymphocyte (CTL) reaction. Body fluidity and The cellular immune response is assisted (Non-Patent Documents 3 and 4). Therefore, CpG ODN is considered to be an immunotherapeutic agent having anti-infectivity, cancer, asthma, and hay fever (Non-Patent Documents 2 and 5).

There are at least four types of CpG ODNs in which the skeleton sequence and the immunostimulating characteristics are different from each other (Non-Patent Document 6). Type D (also known as Type A) CpG ODN typically contains a phosphodiester (PO) backbone and a phosphorothioate (PS) Poly (G) tail (Poly (G) tail), which also contains 1 A palindronic CpG motif, which activates plasma cell-like DCs (pDCs) and produces a large amount of IFN-α, but does not induce pDC maturation or B cell activation (non-patented) Documents 7 and 8). The other three types of ODNs include a PS backbone. K-type (also known as type B) CpG ODN typically contains a plurality of CpG motifs in a non-palindromic structure, which strongly activates B cells to produce IL-6, which activates and matures pDCs, but hardly makes IFN-α production (Non-Patent Documents 8 and 9). In recent years, CpG ODNs of both C and P types have been developed to contain one or two palindrome CpG sequences, both of which can activate B cells as K-type, and can activate pDCs as D-type. However, compared with the P-type CpG ODN, the C-type CpG ODN induces IFN-α production weakly (Non-Patent Documents 10-12). Patent Document 1 describes a plurality of excellent K-type CpG ODNs.

It has been shown that D-type and P-type CpG ODN each form a so-called Hoogsteen base pair and a Watson-Crick base pair high-order structure, which is called G-tetravalent. The homologous 4-strand structure of the Hoogsteen base pair of the G-tetrads, and the Watson-Crick between the cis-palindrome and the trans-palindrome (Watson-Crick) base pair, which is necessary for the production of potent IFN-α by pDCs (Non-Patent Documents 12-14). Such high-order structures are necessary for localization of the initial endosome or by TLR9 signaling, but these are affected by the polymorphism of the product and precipitation, and the results impede its clinical application (non- Patent Document 15). Therefore, only K-type and C-type CpG ODNs can be generally used as immunotherapeutic agents and vaccine adjuvants for humans (Non-Patent Documents 16 and 17). K-type CpG ODN is used in human clinical trials to improve the immunogenicity of vaccines with infectious diseases and cancers (Non-Patent Documents 6 and 16). For the most suitable adjuvant effect, the chemistry between antigen and K-type CpG ODN Sexual and physical connections are necessary. These results show that CpG ODNs of four types (K, D, P, and C) have advantages and disadvantages, and it is expected that "all-in-one" will not agglutinate and activate both B cells and pDCs. Development of CpG ODN.

Schizophyllum commune, Schizophyllum commune, schizophyllan (SPG), a proliferating drug for radiation therapy in patients with cervical cancer, has been approved in Japan for 30 years. Medicine (Non-Patent Document 18). Similarly, lenthinan (LNT), a soluble β-1,3-glucan from Lentinula edodes, was recognized in 1985 as a medicine for patients with inoperable and recurrent gastric cancer. It is used in combination with a fluoropyrimidine-based agent (Non-Patent Documents 19 and 20). It has been shown that β-1,3-glucan forms a complex of polydeoxyadenylic acid (dA) and a triple helix structure (Non-Patent Document 21).

Patent Documents 2 to 4 disclose a gene carrier as a water-soluble complex of β-1,3-glucan and nucleic acid (gene) containing Schizophyllum polysaccharides. the use of. These documents have documented that by forming the complex, the antisense effect of the gene and the tolerance to the nucleic acid decomposing enzyme (nuclease) are enhanced.

Patent Document 5 discloses that by using a polysaccharide having a β-1,3-bond as a carrier (transfection agent), the CpG-containing sequence is increased, and the phosphodiester bond is substituted with a phosphorothioate. The role of an immunostimulatory oligonucleotide linked to a bond or a phosphorodithioate bond.

Patent Document 6 describes an immunostimulatory complex characterized by comprising an immunostimulatory oligonucleotide and a β-1,3-glucose having a long-chain β-1,6-glucoside bond side chain. Glycans.

The present inventors have previously shown that a mouse and a humanized CpG ODN having a phosphodiester bond-binding poly(dA) linked to SPG enhances interleukin production as an influenza vaccine adjuvant or Th2. It acts as a prophylactic or therapeutic agent for cell-associated diseases (Non-Patent Documents 22 and 23, and Patent Document 7). When poly(dA) is added to the 5' end of each of K-type and D-type CpG to form a complex with SPG, the properties of each of K-type and D-form are maintained, and the activity is enhanced. However, achieving high yields of CpG-SPG complexes is difficult to achieve more effective and cost effective preclinical and clinical development. In recent years, it has been shown that when poly(dA) having a phosphorothioate bond is bonded to CpG ODN, the formation of the complex increases to almost 100% (Non-Patent Document 24). However, careful testing to optimize the most suitable humanized CpG sequence and to optimize the "all-in-one" activity of the four types of CpG ODN was not performed.

Patent Document 8 discloses a method for producing an antigen/CpG oligonucleotide/β-1,3-glucan-based ternary complex.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] US 8,030,285 B2

[Patent Document 2] WO 01/034207 A1

[Patent Document 3] WO 02/072152 A1

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2004-107272

[Patent Document 5] WO 2004/100965 A1

[Patent Document 6] Japanese Patent Laid-Open Publication No. 2007-70307

[Patent Document 7] Japanese Patent Laid-Open Publication No. 2008-100919

[Patent Document 8] Japanese Patent Laid-Open Publication No. 2010-174107

[Non-patent literature]

[Non-Patent Document 1] Hemmi, H., et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740-745 (2000).

[Non-Patent Document 2] Krieg, A.M. Therapeutic potential of Toll-like receptor 9 activation. Nature reviews. Drug discovery 5, 471-484 (2006).

[Non-Patent Document 3] Brazolot Millan, C.L., Weeratna, R., Krieg, A.M., Siegrist, C.A. & Davis, H.L. CpG DNA can induce strong Thl humoral and cell-mediated immune responses against hepatitis B surface antigen in young Mice. Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558 (1998).

[Non-Patent Document 4] Chu, RS, Targoni, OS, Krieg, AM, Lehmann, PV & Harding, CV CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. The Journal of experimental medicine 186, 1623- 1631 (1997).

[Non-Patent Document 5] Klinman, D.M. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nature reviews. Immunology 4, 249-258 (2004).

[Non-Patent Document 6] Vollmer, J. & Krieg, A.M. Immunotherapeutic applications of CpG oligodeoxynu cleotide TLR9 agonists. Advanced drug delivery reviews 61, 195-204 (2009).

[Non-Patent Document 7] Krug, A., et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. European journal of immunology 31, 2154-2163 (2001).

[Non-Patent Document 8] Verthelyi, D., Ishii, KJ, Gursel, M., Takeshita, F. & Klinman, DM Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs. Journal of immunology 166, 2372-2377 (2001).

[Non-Patent Document 9] Hartmann, G. & Krieg, A.M. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. Journal of immunology 164, 944-953 (2000).

[Non-Patent Document 10] Hartmann, G., et al. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-alpha induction in plasmacytoid dendritic cells. European journal of immunology 33, 1633-1641 (2003).

[Non-Patent Document 11] Marshall, J.D., et al. Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions. Journal of leukocyte biology 73, 781-792 (2003).

[Non-Patent Document 12] Samulowitz, U., et al. A novel class of immune-stimulatory CpG oligodeoxynucleotides unifies high potency in type I interferon induction with preferred structural properties. Oligonucleotides 20, 93-101 (2010).

[Non-Patent Document 13] Kerkmann, M., et al. Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells. The Journal of biological chemistry 280, 8086-8093 ( 2005).

[Non-Patent Document 14] Klein, D.C., Latz, E., Espevik, T. & Stokke, B.T. Higher order structure of short immunostimulatory oligonucleotides studied by Atomicforce microscopy. Ultramicroscopy 110, 689-693 (2010).

[Non-Patent Document 15] Puig, M., et al. Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates. Nucleic acids research 34, 6488-6495 (2006) .

[Non-Patent Document 16] Bode, C., Zhao, G., Steinhagen, F., Kinjo, T. & Klinman, D.M. CpG DNA as a vaccine adjuvant. Expert review of vaccines 10, 499-511 (2011).

[Non-Patent Document 17] McHutchison, J.G., et al. Phase 1B, randomized, double-blind, dose-escalation trial of CPG 10101 in patients with chronic hepatitis C virus. Hepatology 46, 1341-1349 (2007).

[Non-Patent Document 18] Okamura, K., et al. Clinical evaluation of schizophyllan combined with irradiation in patients with cervical cancer. A randomized controlled study. Cancer 58, 865-872 (1986).

[Non-Patent Document 19] Oba, K.; Kobayashi, M.; Matsui, T.; Kodera, Y.; Sakamoto, J. Individual patient based meta-analysis of lentinan for unresectable/recurrent gastric cancer. Anticancer Res., 2009 , 29, 2739-2746.

[Non-Patent Document 20] Nakano, H.; Namatame, K.; Nemoto, H.; Motohashi, H.; Nishiyama, K.; Kumada, K. A multi-institutional prospective study of lentinan in Advanced gastric cancer patients with unresectable and recurrent diseases: Effect on prolongation of survival and improvement of quality of life. Hepato-Gastroenterol., 1999, 46, 2662-2668.

[Non-Patent Document 21] Sakurai, K., Mizu, M. & Shinkai, S. Polysaccharide-polynucleotide complexes. 2. Complementary polynucleotide mimic behavior of the natural polysaccharide schizophyllan in the macromolecular complex with single-stranded RNA and DNA. Biomacromolecules 2 , 641-650 (2001).

[Non-Patent Document 22] Shimada, N., et al. A polysaccharide carrier to effectively deliver native phosphodiester CpG DNA to antigen-presenting cells. Bioconjugate chemistry 18, 1280-1286 (2007).

[Non-Patent Document 23] Koyama, S., et al. Plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes. Science translational medicine 2, 25ra24 (2010).

[Non-Patent Document 24] Minari, J., et al. Enhanced cytokine secretion from primary macrophages due to Dectin-1 mediated uptake of CpG DNA/beta-1,3-glucan complex. Bioconjugate chemistry 22, 9-15 (2011) .

The problem to be solved by the present invention is to provide a more prior The CpG-SPG complex is a more active immunostimulating agent.

The inventors of the present invention conducted a review and confirmed that a novel complex of K3 (SEQ ID NO: 2) and SPG of K-type CpG ODN having a polydeoxyadenylate tail (poly(dA) tail) at the 3' end was confirmed. That is, K3-SPG, and a novel complex containing K3 (hereinafter referred to as K3-spacer) and SPG having a polydeoxyadenosine tail at the 3' end spacer spacer, that is, K3-spacer-SPG. These form high-grade nanoparticles that are completely soluble. Similarly, the present inventors have also succeeded in producing a novel complex K3-LNT containing the above-mentioned K-type CpG ODN and lentinan (LNT), and a novel complex containing K3-spacer and LNT, namely K3-spacer-LNT. Although K3-SPG, K3-spacer-SPG, K3-LNT, and K3-spacer-LNT do not have a D-type CpG ODN sequence, they also have immunogenic activity specific to K-type CpG ODN (for example, B cells (preferably Human B cells are activated to produce IL-6), and immunogenic activity specific to D-type CpG ODN (for example, activation of plasmacytoid dendritic cells to produce IFN-α). Furthermore, K3-LNT, K3-spacer-LNT, K3-spacer-SPG and K3-SPG have potent vaccine adjuvant activity, and when administered together with antigen, induce antigen-specific humoral immunity and cellularity. Immunizing both, in fact, shows a very strong infection defense effect on RS virus or influenza virus. The present invention has been completed by conducting further reviews based on these knowledge findings.

That is, the present invention is as follows.

[1] An oligodeoxynucleotide comprising an oligodeoxynucleotide containing a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenosine, wherein The oxyadenylate is bound to the 3' side of the humanized K-type CpG oligodeoxynucleotide by spacer.

[2] The oligodeoxynucleotide according to [1], wherein the humanized K-type CpG oligodeoxynucleotide is 10 nucleotides or more in length, and comprises 1 or a plurality of unmethylated CpG motifs. body.

[3] The oligodeoxynucleotide according to [1] or [2] wherein the humanized K-type CpG oligodeoxynucleotide comprises the sequence TCGA or TCGT.

[4] The oligodeoxynucleotide according to any one of [1] to [3] wherein the humanized K-type CpG oligodeoxynucleotide comprises the nucleotide sequence represented by SEQ ID NO: 1.

[5] The oligodeoxynucleotide according to any one of [1] to [4] wherein a part or all of a phosphodiester bond of the oligodeoxynucleotide is substituted with a phosphorothioate bond.

[6] The oligodeoxynucleotide according to [5], wherein all of the phosphodiester bond of the oligodeoxynucleotide is substituted with a phosphorothioate bond.

[7] The oligodeoxynucleotide according to any one of [1] to [6] wherein the polydeoxyadenosine is 20 to 60 nucleotides in length.

[8] The oligodeoxynucleotide according to any one of [1] to [7] wherein the spacer is of the formula (A)

(wherein m is 2, 3, 4, 5, 6, 7, 8, or 9, n is 1, 2, 3, 4, 5 or 6, and formula (3m+2)n is 8 or more and 60 or less, plural a group represented by R ¹ , R ² , R ³ and R ⁴ independently of each other as a hydrogen atom or a C _1-6 alkyl group, X is an oxygen atom or a sulfur atom, or a formula (B)

(where s is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, t is 1, 2, 3, 4, 5 or 6, and (s+3)t is 8 In the above 60 or less, the plural R ⁵ and R ⁶ are each independently a hydrogen atom or a C _1-6 alkyl group, and X is an oxygen atom or a sulfur atom.

[9] The oligodeoxynucleotide according to any one of [1] to [8] wherein the spacer is of the formula (A1)

(wherein m is 4, 5 or 6, n is 1 or 2, and the formula (3m+2)n is 14 or more and 40 or less, and the plural R ¹ , R ² , R ³ and R ^{4 are} all a hydrogen atom, X a group represented by a sulfur atom) or a formula (B1)

(wherein s is 8, 9, 10, 11 or 12, t is 1 or 2, (s+3)t is 14 or more and 40 or less, and plural R ⁵ and R ^{6 are} all hydrogen atoms, and X is a sulfur atom. ) the base represented.

[10] The oligodeoxynucleotide according to any one of [1] to [9] wherein the spacer is

A group represented by either of them or a group in which the group is bonded 2 or 3 times.

[11] A complex comprising the oligodeoxynucleotide according to any one of [1] to [10], and β-1,3-glucan.

[12] The complex according to [11], wherein the β-1,3-glucan is lentinan, schizophyllum polysaccharide, scleroglucan, and Catrando. Curdlan, pachyman, grifolan, or laminaran.

[13] The complex according to [12], wherein the β-1,3-glucan is lentinan, schizophyllum polysaccharide or scleroglucan.

[14] The complex according to [11], which comprises the oligodeoxynucleotide described in (i) below, and the β-1,3-glucan described in (ii), (i) in the inclusion sequence The 3' side spacer spacer of the oligodeoxynucleotide of the nucleotide sequence represented by the number 1 is combined with the polydeoxyadenosine of 20 to 60 nucleotides in length, and the entire phosphodiester bond An oligodeoxynucleotide substituted with a phosphorothioate linkage; (ii) a lentinan or a Schizophyllum polysaccharide.

[15] The complex according to [11], which comprises "on the 3' side of the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, a length of 20 to 60 nucleotides Deoxyadenosine compartment contains the following formula

a base represented by either of them or repeating the base 2 or 3 times a oligodeoxynucleotide in which all of the phosphodiester bonds are substituted by a phosphorothioate bond, and "β-1,3-glucan (for example, lentinan, Schizophyllum polysaccharides).

[16] The complex according to [15], which comprises "on the 3' side of the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, and is 30 to 50 nucleotides in length. Oxygen adenosine spacers include the following formula

a group represented by either of them, or an oligodeoxynucleotide in which all of the phosphodiester bonds are substituted by a phosphorothioate bond, in which the group is repeated 2 or 3 times. And "β-1,3-glucan".

[17] The complex according to [16], which comprises "on the 3' side of the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, a length of 30 to 45 nucleotides Deoxyadenosine compartment contains the following formula

a group represented by either of them, or an oligodeoxynucleotide in which all of the phosphodiester bonds are substituted by a phosphorothioate bond, in which the group is repeated 2 or 3 times. And "β-1,3-glucan".

[18] The complex according to [17], which comprises "a 3' side of an oligodeoxynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 1, a polydeoxygen of 40 nucleotides in length Adenylate spacer

Any one of the oligodeoxynucleotides in which the base group is repeated 2 or 3 times and the bonded group is bonded, and the phosphodiester bond is replaced by a phosphorothioate bond, and the mushroom Polysaccharide or Schizophyllum polysaccharides.

[19] The composite according to any one of [11] to [18] which is a triple helix structure.

[20] The complex according to any one of [11] to [19] which has an activity of activating B cells to produce IL-6, and an activation of dendritic cells to produce IFN-? active.

[21] A pharmaceutical composition comprising the oligodeoxynucleotide according to any one of [1] to [10], or the complex according to any one of [11] to [20].

[22] The pharmaceutical composition according to [21], which is for use in the prevention or treatment of viral infection, cancer, allergic disease, intracellular parasitic protozoa or bacterial infection.

[23] The pharmaceutical composition according to [22], which is for use in the prevention or treatment of a viral infection.

[24] The pharmaceutical composition according to [23], wherein the viral infection is an RS virus or an influenza virus infection.

[25] A type I and/or type II interferon production-inducing agent comprising the complex according to any one of [11] to [20].

[26] An immunostimulating agent comprising the oligodeoxynucleotide according to any one of [1] to [10], or the complex according to any one of [11] to [20].

[27] The immunostimulating agent according to [26], which is a vaccine adjuvant.

[28] A prophylactic or therapeutic agent for a viral infection, a cancer, an allergic disease, an intracellular parasitic protozoan or a bacterial infection, which comprises the oligodeoxygen core according to any one of [1] to [10] A glycoside or a complex according to any one of [11] to [20].

[29] The prophylactic or therapeutic agent according to [28], wherein the viral infection is an RS virus or an influenza virus infection.

[30] The use of the oligodeoxynucleotide according to any one of [1] to [10] or the composition according to any one of [11] to [20], which is for use in a pharmaceutical composition Manufacturing.

[31] The use according to [30], wherein the pharmaceutical composition is a pharmaceutical composition for prevention or treatment of viral infection, cancer, allergic disease, intracellular parasitic protozoa or bacterial infection.

[32] The use of [31], wherein the viral infection is an RS virus or an influenza virus infection.

[33] A method for the treatment or prevention of a disease in a warm-blooded animal The oligodeoxynucleotide according to any one of [1] to [10], or the complex according to any one of [11] to [20], which is administered in a pharmacologically effective amount. To the warm-blooded animal.

[34] The method according to [33], wherein the disease is a viral infection, a cancer, an allergic disease, an intracellular parasitic protozoa or a bacterial infection.

[35] The method according to [34], wherein the viral infection is an RS virus or an influenza virus infection.

[36] The method according to any one of [33] to [35] wherein the warm-blooded animal is a human.

[37] A method for inducing a defense immune response in a warm-blooded animal, comprising a pharmacologically effective amount of the oligodeoxynucleotide according to any one of [1] to [10], or as [11] The complex described in any one of [20] is administered to the warm-blooded animal.

[38] The oligodeoxynucleotide according to any one of [1] to [10], wherein the complex according to any one of [11] to [20] is used for a viral infection, Treatment or prevention of cancer, allergic diseases, intracellular parasitic protozoa or bacterial infections.

[39] The oligodeoxynucleotide or complex according to [38], wherein the viral infection is an RS virus or an influenza virus infection.

[40] A pharmaceutical composition comprising: (a) the oligodeoxynucleotide according to any one of [1] to [10], or the method according to any one of [11] to [20] Complex, and (b) antigen.

[41] The composition according to [40], which is for inducing an immune response against the antigen.

[42] The composition according to [41], wherein the antigen is an antigen derived from a pathogen.

[43] The composition according to [42], which is for the prevention or treatment of an infectious disease of a pathogen.

[44] The composition according to [43], wherein the pathogen is a virus.

[45] The composition according to [44], wherein the virus is an RS virus or an influenza virus.

According to the present invention, an oligodeoxynucleotide having excellent immunostimulating activity or a complex comprising the same can be provided. In particular, the complex of the present invention has both an immunostimulating activity specific to K-type CpG ODN and an immunostimulating activity specific to D-type CpG ODN. Furthermore, the complex of the present invention has potent vaccine adjuvant activity, and when the complex of the present invention is vaccinated together with an antigen, it stimulates both antigen-specific humoral immunity and cellular immunity, and shows very strong Infected defense effect. Thus, the composite system of the invention is useful as an immunostimulant or vaccine adjuvant.

[Fig. 1] Pan-IFN-a inducing ability and IL-6 inducing ability of K3-spacer alone, K3-spacer-LNT complex and K3-SPG complex in human PBMC. K3-spacer-alone: K3-(S18)-dA40, K3-(S18)2-dA40, K3-(S18)3-dA40. K3-spacer-LNT complex: K3-(S18)-dA40-LNT#1/#2, K3-(S18)2-dA40-LNT#1/#2, K3-(S18)3-dA40-LNT# 1/#2 (#1, #2 are each made with mG/dA molar ratio of 2.5 and 3.0). K3-SPG complex: K3-SPG. NC: Borrow From the untreated negative control group.

[Fig. 2] The RSV F antigen-specific IgG antibody titer in the serum of mice inoculated with an adjuvant-added RSV F subunit vaccine. K3-spacer-addition group: F+K3-(S18)-dA40, F+K3-(S18)2-dA40, F+K3-(S18)3-dA40. K3-spacer-LNT complex addition group: F+K3-(S18)-dA40-LNT, F+K3-(S18)2-dA40-LNT, F+K3-(S18)3-dA40-LNT. K3-SPG complex addition group: F+K3-SPG. Alum phosphate addition group: F+Alum. RSV F antigen alone: F. NC: by untreated negative control group.

[Fig. 3] Interleukin-inducing ability specifically induced by RSV F antigen stimulation in mice inoculated with an adjuvant-added RSV F subunit vaccine. K3-spacer-addition group: F+K3-(S18)-dA40, F+K3-(S18)2-dA40, F+K3-(S18)3-dA40. K3-spacer-LNT complex addition group: F+K3-(S18)-dA40-LNT, F+K3-(S18)2-dA40-LNT, F+K3-(S18)3-dA40-LNT. K3-SPG complex addition group: F+K3-SPG. Alum phosphate addition group: F+Alum. RSV F antigen alone: F. NC: by untreated negative control group.

[Fig. 4] Pan-IFN-a inducing ability and IL-6 inducing ability of K3-spacer-LNT complex and K3-SPG complex in human PBMC. K3-spacer-LNT complex: K3-(S18)2-dA30-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)2-dA40- LNT, K3-(S12)3-dA40-LNT, K3-(C12)2-dA40-LNT, K3-(C12)3-dA40-LNT. K3-SPG complex: K3-SPG. NC: by untreated negative control group.

[Fig. 5] Mice inoculated with adjuvant-added RSV F subunit vaccine The RSV F antigen-specific IgG antibody titer in the serum. K3-spacer-LNT complex addition group: F+K3-(S18)2-dA30-LNT, F+K3-(S18)2-dA35-LNT, F+K3-(S18)2-dA40-LNT, F +K3-(S12)2-dA40-LNT, F+K3-(S12)3-dA40-LNT, F+K3-(C12)2-dA40-LNT, F+K3-(C12)3-dA40-LNT . K3-SPG complex addition group: F+K3-SPG. Alum phosphate addition group: F+Alum. RSV F antigen alone: F. NC: by untreated negative control group.

[Fig. 6] Interleukin-inducing ability specifically induced by RSV F antigen stimulation in mice inoculated with an adjuvant-added RSV F subunit vaccine. K3-spacer-LNT complex addition group: F+K3-(S18)2-dA30-LNT, F+K3-(S18)2-dA35-LNT, F+K3-(S18)2-dA40-LNT, F +K3-(S12)2-dA40-LNT, F+K3-(S12)3-dA40-LNT, F+K3-(C12)2-dA40-LNT, F+K3-(C12)3-dA40-LNT . K3-SPG complex addition group: F+K3-SPG. Alum phosphate addition group: F+Alum. RSV F antigen alone: F. NC: by untreated negative control group.

[Fig. 7] Pan-IFN-a inducing ability and IL-6 inducing ability of K3-spacer-LNT complex and K3-SPG complex in human PBMC. K3-spacer-LNT complex: K3-(S18)-dA35-LNT, K3-(S18)-dA40-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)-dA35-LNT, K3-(S12)-dA40-LNT, K3-(S12)2-dA35-LNT, K3-(S12)2-dA40-LNT, K3-(S9)-dA35- LNT, K3-(S9)-dA40-LNT, K3-(S9)2-dA35-LNT, K3-(S9)2-dA40-LNT. K3-SPG complex: K3-SPG. NC: by untreated negative control group.

[Fig. 8] Mice vaccinated with adjuvant-added RSV F subunit vaccine The RSV F antigen-specific IgG antibody titer in the serum. K3-spacer-LNT complex addition group: F+K3-(S18)-dA35-LNT, F+K3-(S18)-dA40-LNT, F+K3-(S18)2-dA35-LNT, F+K3 -(S18)2-dA40-LNT, F+K3-(S12)-dA35-LNT, F+K3-(S12)-dA40-LNT, F+K3-(S12)2-dA35-LNT, F+K3 -(S12)2-dA40-LNT, F+K3-(S9)-dA35-LNT, F+K3-(S9)-dA40-LNT, F+K3-(S9)2-dA35-LNT, F+K3 - (S9) 2-dA40-LNT. Alum phosphate addition group: F+Alum. RSV F antigen alone: F. NC: by untreated negative control group.

[Fig. 9] Interleukin-inducing ability specifically induced by RSV F antigen stimulation in mice inoculated with an adjuvant-added RSV F subunit vaccine. K3-spacer-LNT complex addition group: F+K3-(S18)-dA35-LNT, F+K3-(S18)-dA40-LNT, F+K3-(S18)2-dA35-LNT, F+K3 -(S18)2-dA40-LNT, F+K3-(S12)-dA35-LNT, F+K3-(S12)-dA40-LNT, F+K3-(S12)2-dA35-LNT, F+K3 -(S12)2-dA40-LNT, F+K3-(S9)-dA35-LNT, F+K3-(S9)-dA40-LNT, F+K3-(S9)2-dA35-LNT, F+K3 - (S9) 2-dA40-LNT. Alum phosphate addition group: F+Alum. RSV F antigen alone: F. NC: by untreated negative control group.

[Fig. 10] A method of complexing a CpG ODN having a spacer with LNT or SPG.

[Fig. 11] Pan-IFN-a inducing ability and IL-6 inducing ability of the K3-spacer-SPG complex in human PBMC. K3-spacer-SPG complex: K3-(S9)-dA40-SPG, K3-(S9)2-dA40-SPG, K3-(S12)-dA40-SPG, K3-(S12)2-dA40-SPG, K3-(S18)-dA40-SPG K3-(S18)2-dA40-SPG, K3-(C12)2-dA40-SPG, K3-(C12)3-dA40-SPG. NC: by untreated negative control group.

[Fig. 12] RSV F antigen-specific IgG antibody titer in serum of mice inoculated with an adjuvant-added RSV F subunit vaccine. K3-spacer-SPG complex addition group: F+K3-(S9)-dA40-SPG, F+K3-(S9)2-dA40-SPG, F+K3-(S12)-dA40-SPG, F+K3 -(S12)2-dA40-SPG, F+K3-(S18)-dA40-SPG, F+K3-(S18)2-dA40-SPG, F+K3-(C12)2-dA40-SPG, F+ K3-(C12)3-dA40-SPG. RSV F antigen alone: F. NC: by untreated negative control group.

[Fig. 13] Interleukin-inducing ability specifically induced by RSV F antigen stimulation in mice inoculated with an adjuvant-added RSV F subunit vaccine. K3-spacer-SPG complex addition group: F+K3-(S9)-dA40-SPG, F+K3-(S9)2-dA40-SPG, F+K3-(S12)-dA40-SPG, F+K3 -(S12)2-dA40-SPG, F+K3-(S18)-dA40-SPG, F+K3-(S18)2-dA40-SPG, F+K3-(C12)2-dA40-SPG, F+ K3-(C12)3-dA40-SPG. RSV F antigen alone: F. NC: by untreated negative control group. Non-sti: No irritation.

[Formation of the Invention]

Oligo-deoxynucleotide

The present invention provides an oligodeoxynucleotide (hereinafter, referred to as an oligodeoxynucleotide of the present invention) comprising a K-type CpG oligodeoxynucleotide and polydeoxyadenylate (dA).

The oligodeoxynucleotide of the present invention comprises a phosphodiester bond modified (for example, some or all of the phosphodiester bond is substituted by a phosphorothioate bond) By.

The oligodeoxynucleotide of the invention comprises a pharmaceutically acceptable salt.

Further, in the present specification, an oligodeoxynucleotide is synonymous with an ODN system. Also, the presence or absence of "humanized K-type CpG oligodeoxynucleotides (CpG ODN)" and "humanized K-type CpG oligodeoxynucleotides (CpG ODN) residues" and the term "residues" at the end Irrelevant and synonymous, interchangeable. Furthermore, polydeoxyadenylate is synonymous with polydeoxyadenosine (residue). The term "residue" means a partial structure of a compound having a relatively large molecular weight, but in the present specification, "humanized K-type CpG oligodeoxynucleotide (CpG ODN)" means an independent molecule or a larger molecular weight. The partial structure of the compound can be easily understood by the context if it is a person having ordinary knowledge in the art. The terms related to the other partial structures contained in the oligodeoxynucleotide of the present invention are also the same for "polydeoxyadenosine" and the like.

CpG oligodeoxynucleotide (CpG ODN) is a single-stranded DNA containing an immunologically active unmethylated CpG motif and is an agonist of TLR9. CpG ODN has four types of K-type (also known as B-type), D-type (also known as A-type), C-type and P-type, which have different skeleton sequences and immunosynthesis characteristics (Advanced drug delivery reviews 61, 195-204). (2009)). The oligodeoxynucleotide of the present invention comprises the K-type CpG ODN in these.

The K-type CpG ODN has a so-called "unmethylated CpG motif that typically contains a non-palindromic structure, which activates B cells to produce IL-6, but hardly induces plasmacytoid dendritic cells (pDCs). CpG ODN of the structure and function of IFN-α production. The unmethylated CpG model system refers to a short nucleotide sequence containing at least one cytosine (C)-guanine (G) sequence, and the cytosine in the cytosine-guanine sequence The fifth place was not methylated. Further, in the following description, CpG means unmethylated CpG unless otherwise specified. Therefore, the oligodeoxynucleotide of the present invention has an immunostimulating activity specific to K-type CpG ODN by including a K-type CpG ODN (for example, activating B cells (preferably human B cells) to make IL -6 produced activity). In this technical field, most humanized K-type CpG ODNs are known (Journal of Immunology 166, 2372-2377 (2001); Journal of Immunology 164, 944-953 (2000); US 8,030, 285 B2).

The K-type CpG ODN contained in the oligodeoxynucleotide of the present invention is preferably humanized. "Humanized" means having an agonist activity against human TLR9. Therefore, the oligodeoxynucleotide of the present invention comprising a humanized K-type CpG ODN has an immunostimulatory activity specific to a human type K CpG ODN (for example, an activity of activating human B cells to produce IL-6) .

The K-type CpG ODN suitable for use in the present invention is 10 nucleotides or more in length and contains one or a plurality of unmethylated CpG motifs.

The K-type CpG ODNs more suitable for use in the present invention contain a non-palindromic structure comprising one or a plurality of CpG motifs. A more suitable K-type CpG ODN is composed of a non-palindromic structure comprising one or a plurality of CpG motifs.

The humanized K-type CpG ODN is generally characterized by a 4-base CpG motif composed of TCGA or TCGT. Further, in most cases, two or three such 4 base CpG motifs are contained in one humanized K-type CpG ODN. Therefore, in a preferred aspect, the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention comprises at least one, more preferably 2 or more, further preferably 2 or 3 by TCGA or TCGT. 4 bases CpG motif. In the case where the K-type CpG ODN has 2 or 3 4 base CpG motifs, the 4 base CpG motifs may be the same or different. However, it is not particularly limited as long as it has agonist activity against human TLR9.

More preferably, the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention comprises the nucleotide sequence represented by SEQ ID NO: 1.

The length of the K-type CpG ODN is not included as long as the oligodeoxynucleotide of the present invention has immunostimulating activity (for example, activation of B cells (preferably human B cells) to produce IL-6) It is particularly limited, but is preferably 100 nucleotides or less in length (for example, 10-75 nucleotides in length). The length of the K-type CpG ODN is preferably less than 50 nucleotides in length (e.g., 10-40 nucleotides in length). The length of the K-type CpG ODN is more preferably 30 nucleotides or less (for example, 10-25 nucleotides in length). The length of the K-type CpG ODN is preferably 12-25 nucleotides in length.

The length of the polydeoxyadenylate (dA) is not a sufficient length for forming a triple helix structure together with the β-1,3-glucan (preferably lentinan or schizophyllum) chain. It is particularly limited, but from the viewpoint of forming a stable triple helix structure, it is usually 20 nucleotides or longer, preferably 40 nucleotides or longer, and more preferably 60 nucleotides or longer. Poly- adenosine monophosphate (poly dA) can form a stable triple helix structure with β-1,3-glucan as it grows longer. Therefore, there is no theoretical limit, but when it is too long, it becomes oligodeoxygenation. The reason for the variation in the length of the nucleotide synthesis is usually 100 nucleotides or less, preferably 80 or less. On the other hand, in addition to the formation of the aforementioned stable triple helix structure, the present invention is combined with β-1,3-glucan per unit amount. The length of the deoxyadenosine is preferably 20 to 60 nucleotides from the viewpoint of avoidance of the deviation of the length of the oligodeoxynucleotide and the length of the synthesis of the oligodeoxynucleotide. Length (specifically, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides in length), more preferably 30 to 50 Nucleotide length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleosides Acid length), preferably 30 to 45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleosides) Acid length). In particular, a case of more than 30 nucleotides in length shows good complexing efficiency. The oligodeoxynucleotide of the present invention has an activity of forming a triple helix structure together with a double-stranded Schizophyllum polysaccharide chain by containing polydeoxyadenosine. Further, there is a case where polydeoxyadenosine is labeled as "poly(dA)" or "poly(dA)".

The oligodeoxynucleotide of the present invention may comprise a plurality of K-type CpG ODNs and/or polydeoxyadenosines, but preferably K-type CpG ODNs and polydeoxyadenosines are contained. One of them is preferably composed of one each of K-type CpG ODN and polydeoxyadenosine.

The oligodeoxynucleotide of the present invention is characterized in that polydeoxyadenylate is disposed on the 3' side of the K-type CpG ODN. By this configuration, the complex of the present invention (details as described below) has an immunostimulating activity specific to the D-type CpG ODN in addition to the immunostimulating activity specific to the K-type CpG ODN.

In the oligodeoxynucleotide of the present invention, the K-type CpG ODN and the polydeoxyadenosine are linked by a spacer. In the present invention, the spacer is a group in which 8 to 60 atoms are connected in a straight line. The atom to be used as the spacer in the present invention is not particularly limited as long as it is a linearly linked atom. For example, carbon is exemplified. The atom, the nitrogen atom, the oxygen atom, the halogen atom, the phosphorus atom, and the sulfur atom are preferably a carbon atom, an oxygen atom or a phosphorus atom. The atom of the linear chain forming the spacer used in the present invention may be bonded to a substituent of a hydrogen atom, an oxygen atom, a sulfur atom, a hydroxyl group, a C _1-6 alkyl group or the like, and the substituent is preferably a hydrogen atom or sulfur. An atom, a hydroxyl group or a methyl group is more preferably a hydrogen atom, a sulfur atom or a hydroxyl group. The spacer used in the present invention is more preferably the formula (A)

(wherein m is 2, 3, 4, 5, 6, 7, 8 or 9 (preferably 4, 5 or 6), and n is 1, 2, 3, 4, 5 or 6 (preferably 1) Or 2), the formula (3m+2)n is 8 or more and 60 or less (preferably 14 or more and 40 or less), and the plural R ¹ , R ² , R ³ and R ⁴ are each independently a hydrogen atom or C _1-6 An alkyl group (preferably all of which is a hydrogen atom), X is a group represented by an oxygen atom or a sulfur atom (preferably a sulfur atom), or a formula (B)

(wherein s is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 (preferably 8, 9, 10, 11 or 12), and t is 1, 2, 3, 4, 5 or 6 (preferably 1 or 2), (s+3)t is 8 or more and 60 or less (preferably 14 or more and 40 or less), and the plural R ⁵ and R ⁶ are each independently a hydrogen atom or C. _a group represented by _1-6 alkyl groups (preferably all of them are hydrogen atoms), and X is an oxygen atom or a sulfur atom (preferably a sulfur atom), and more preferably a formula

A group represented by either of the groups or a group in which the group is bonded 2 or 3 times (preferably 2 times). Here, the basis of the bonding is repeated twice, for example, the following formula

The base represented.

The spacer used in the present invention can be produced, for example, using the corresponding amidite. For the sulfonamide of the spacer represented by the formula (A), for example, PEG sulfonamide (a commercially available product or a method described in JP-A-2002-176987) can be used. The sulfonamide corresponding to the spacer represented by the formula (B) can be obtained, for example, by converting an alkanediol having a hydroxyl group at both terminals to a decylamine according to the method described in WO99/19474.

The oligodeoxynucleotides of the present invention can be produced, for example, by the method described in Nucleic Acids in Chemistry and Biology, 3. Chemical synthesis (1990) ed. G. Michael Blackburn and Michael J. Gait. Oxford University Press, in particular , at the 5' position of the polydeoxyadenylate of dA30, dA35, dA40, etc., the atmidamine corresponding to the above spacer is coupled n times, and then, by making the corresponding K-type CpG oligodeoxynucleotide Partially coupled and manufactured.

In a preferred aspect, the oligodeoxynucleotide of the present invention comprises a K-type CpG ODN (specifically, for example, an oligodeoxynucleotide comprising a nucleotide sequence represented by SEQ ID NO: 1), a spacer And polydeoxyadenosine, K-type CpG ODN is located at the 5' end of the oligodeoxynucleotide, polydeoxygen gland The glucosinolate is located at the 3' end, and the K-type CpG ODN is linked to the polydeoxyadenosine based on a spacer. Specifically, it is based on the 3' end of the oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, and is 20 to 60 nucleotides in length (more preferably 30 to 50 nucleosides). Acid length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length) , preferably 30 to 45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 nucleotides in length) a polydeoxynucleotide to which a polydeoxyadenosine mediates a spacer. For example, the oligodeoxynucleotides listed in the table below.

The nucleotide sequences (removal of the spacer portion) of the oligodeoxynucleotides enumerated in the above table are shown in SEQ ID NO: 2 to 17.

The full length of the oligodeoxynucleotide of the present invention (the total length of the nucleotides other than the spacer) is usually 30 to 200 nucleotides in length, preferably 35 to 100 nucleotides in length, more preferably 40 to 80 nucleotides in length (specifically, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides in length), more preferably 50 to 70 nucleotides in length (specifically, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nucleotides in length, optimally 50 to 65 nucleotides in length (specifically, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 nucleotides in length).

The oligodeoxynucleotide of the present invention can be suitably modified to be resistant to decomposition in vivo (for example, by exonuclease or endonuclease). Preferably, the alteration comprises a phosphorothioate modification or a phosphorodithioate modification. That is, a part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is substituted with a phosphorothioate bond or a phosphorodithioate bond.

Preferably, the oligodeoxynucleotide of the present invention comprises a modification of a phosphodiester bond, more preferably a modification of a phosphodiester bond to a phosphorothioate linkage (ie, as described in WO 95/26204, non-crosslinked oxygen) One of the atoms is replaced by a sulfur atom). That is, part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is substituted with a phosphorothioate bond.

The oligodeoxynucleotide of the present invention is preferably a K-type CpG ODN, comprising a modification using a phosphorothioate bond or a phosphorodithioate bond, more preferably all of the phosphodiester bond of the K-type CpG ODN. It is substituted with a phosphorothioate bond. Further, the oligodeoxynucleotide of the present invention is preferably a polydeoxyadenylate comprising a phosphorothioate bond or a phosphorodithioate bond, more preferably a phosphate of the polydeoxyadenosine. All of the ester bonds are replaced by sulfur Phospholipid bond. More preferably, all of the phosphodiester bonds of the oligodeoxynucleotides comprising the humanized K-type CpG oligodeoxynucleotide and polydeoxyadenosine are substituted with phosphorothioate linkages. Preferably, the oligodeoxynucleotide of the invention is at the 3' end of a humanized K-type CpG oligodeoxynucleotide (eg, SEQ ID NO: 1), which is 20 to 60 nucleotides in length (better) 30 to 50 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length, optimally 30 to 45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or a 45-nucleotide length) polydeoxyadenosine, an oligodeoxynucleotide that binds to a spacer, and all of the phosphodiester linkages contained in the oligodeoxynucleotide are replaced by Phosphorothioate linkage. In the oligodeoxynucleotide of the present invention, it is expected that the anti-decomposition resistance can be enhanced by the phosphorothioate bond, and the immunostimulating activity can be expected (for example, activation of B cells to produce IL-6) Enhancement of activity) and high yield of CpG-β-1,3-glucan complex. Further, the phosphorothioate linkage in the present specification is synonymous with a phosphorothioate backbone, and the phosphodiester linkage is synonymous with a phosphoric acid backbone.

The oligodeoxynucleotide of the present invention comprises all pharmaceutically acceptable salts, esters, or salts of such esters of the above oligodeoxynucleotides.

The pharmaceutically acceptable salt of the oligodeoxynucleotide of the present invention is preferably an alkali metal salt such as a sodium salt, a potassium salt or a lithium salt; an alkaline earth metal salt such as a calcium salt or a magnesium salt. a metal salt of an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickel salt, a cobalt salt or the like; an inorganic salt such as an ammonium salt; for example, t-octylamine salt, dibenzylamine salt, porphyrin salt, glucosamine Salt, alkyl phenylglycine, ethylenediamine salt, N-methyl reduced glucosamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine An amine salt of a salt, a tetramethylammonium salt, an organic salt of a hydroxymethylaminomethane salt; a halogen atom, such as a hydrofluoride, a hydrochloride, a hydrobromide or a hydroiodide An acid salt of a nitrate, a perchlorate, a sulfate, a phosphate, or the like; a lower alkanesulfonate such as a methanesulfonate, a trifluoromethanesulfonate or an ethanesulfonate; Acid salt, aryl sulfonate of p-tosylate; acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleic acid An organic acid salt of a salt or the like; and an amine acid salt such as a glycinate, an amidate, a arginine, an alanate, a glutamate or an aspartate. As a more preferable salt, an alkali metal salt such as a sodium salt, a potassium salt or a lithium salt; and, for example, t-octylamine salt, dibenzylamine salt, porphyrin salt, glucosamine salt, phenylglycol Alkylamine salt, ethylenediamine salt, N-methyl reduced glucosamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzyl Ethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine An amine salt of a salt, a tetramethylammonium salt, an organic salt of a hydroxymethylaminomethane salt or the like. Further preferred salts include sodium salts, potassium salts, lithium salts, triethylamine salts and hydroxymethylaminomethane salts.

The oligodeoxynucleotide of the present invention may also exist as a hydrate, and the hydrate as described above is also included in the present invention.

The oligodeoxynucleotide of the present invention may be in the form of a single strand, a double strand, or a triple strand, but is preferably a single strand.

The oligodeoxynucleotide of the present invention is present based on a molecule The case of optical isomers in the asymmetric center. The oligodeoxynucleotides of the present invention, such isomers and mixtures of such isomers in any ratio are included in the inventors unless otherwise specified.

The oligodeoxynucleotides of the invention are preferably isolated. "Isolation" means the operation of removing a factor other than the intended component, and is detached from the naturally occurring state. The purity of the "isolated oligodeoxynucleotide" (the percentage of the weight of the desired oligodeoxynucleotide to the total weight of the object to be evaluated) is usually 70% or more, preferably 80% or more, more preferably More than 90%, further preferably more than 99%.

The oligodeoxynucleotide of the present invention is useful as an immunostimulating agent because it has excellent immunostimulating activity (for example, activation of B cells (preferably human B cells) to produce IL-6). . Furthermore, the oligodeoxynucleotide of the present invention has a triple helix structure together with a double-stranded β-1,3-glucan (preferably lentinan, schizophyllum, or scleroglucan). The nature of the invention is such that it is used in the modulation of the composite of the invention described below.

2. Complex

The present invention provides a complex comprising the above-described oligodeoxynucleotide of the present invention and β-1,3-glucan (hereinafter referred to as a complex of the present invention).

The above-mentioned oligodeoxynucleotide of the present invention contains K-type CpG ODN, and it exerts an immunostimulating activity specific to K-type CpG ODN alone (for example, activation of B cells (preferably human B cells) to cause IL -6 produced activity), lacking the immunostimulating activity specific to D-type CpG ODN (for example, activation of plasmacytoid dendritic cells to produce IFN-α). However, surprisingly, by using β-1,3-glucan (preferably shiitake mushrooms) The polysaccharide or Schizophyllum polysaccharide forms a complex, and does not require the sequence of the D-type CpG ODN to obtain the immunostimulating activity specific to the D-type CpG ODN (for example, the activation of plasmacytoid dendritic cells to produce IFN-α) ). That is, the complex of the present invention has an immunostimulating activity specific to K-type CpG ODN (for example, an activity of activating B cells (preferably human B cells) to produce IL-6), and a D-type CpG ODN-specific one. The immunostimulating activity (for example, the activity of plasmin-like dendritic cells (preferably human plasmacytoid dendritic cells) to activate IFN-α).

Examples of the β-1,3-glucan used in the present invention include lentinan, schistosomiasis, scleroglucan, carterin, lycium polysaccharide, ash tree flower polysaccharide, and lenbutose. The β-1,3-glucan preferably contains a large amount of 1,6-glucopyranoside branches such as lentinan, schizophyllum polysaccharide or scleroglucan (side chain ratio 33 to 40%). The β-1,3-glucan is more preferably lentinan or Schizophyllum polysaccharide, and the best is lentinan.

Lentinus edodes polysaccharide (LNT) is a well-known β-1,3-1,6-glucan from shiitake mushroom, and has a molecular formula of (C ₆ H ₁₀ O ₅ )n and a molecular weight of about 30 to 700,000. Almost insoluble in water, methanol, ethanol (95), or acetone, but dissolved in DMSO or aqueous sodium hydroxide solution of polar organic solvent.

Lentinus edodes has enhanced activity of activated macrophages, killer T cells, natural killer cells and antibody-dependent macrophage vector cytotoxicity (ADMC) (Hamuro, J., et al.: Immunology, 39, 551-559). , 1980, Hamuro, J., et al.: Int. J. Immunopharmacol., 2, 171, 1980, Herlyn, D., et al.: Gann, 76, 37-42, 1985). In animal experiments, it is considered to be the same system Synthetic tumors and autologous tumors are administered in combination with chemotherapeutic agents, and have tumor growth inhibiting effects and prolonged effects. Further, even if it is administered alone by lentinan, it is considered to have a tumor growth inhibiting effect and a prolonged effect. In clinical trials, patients with inoperable or recurrent gastric cancer are considered to have a prolonged survival period by oral administration with tegafur (Pharmaceutical Interview Form) It is approved in Japan with 1mg "Ajinomoto"). The effect caused by the separate administration of lentinan has not been confirmed at present.

Schizophyllum polysaccharide (SPG) is a well-known soluble β-glucan from Schizophyllum. SPG contains the main chain of β-(1→3)-D-glucan and one β-(1→6)-D-glucosyl side chain (Tabata) of each of three glucoses. , K., Ito, W., Kojima, T., Kawabata, S. and Misaki A., "Carbohydr. Res.", 1981, 89, 1, p. 121-135). SPG is used as a clinical drug for intramuscular injection of an immunoenhancement method for gynecological cancer for more than 20 years (Shimizu, Chen, Hoji, Zengyuan, "Biotherapy", 1990, 4, p.1390, Hasegawa, "Oncology And Chemotherapy", 1992, 8, p. 225), safety in vivo has been confirmed (Theresa, M. McIntire and David, A. Brant, "J, Am. Chem. Soc.", 1998, 120, P.6909).

In the present specification, "complex" means a product obtained by combining a plurality of molecules by a non-covalent bond or a covalent bond such as electrostatic bonding, vanadium bond, hydrogen bond, hydrophobic interaction or the like.

The complex of the present invention can form a pharmaceutically acceptable salt, and as such a salt, an alkali metal salt such as a sodium salt, a potassium salt or a lithium salt; an alkaline earth metal salt such as a calcium salt or a magnesium salt is preferable. a metal salt of an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickel salt, a cobalt salt or the like; an inorganic salt such as an ammonium salt; for example, t-octylamine salt, dibenzylamine salt, porphyrin salt, glucosamine Salt, alkyl phenylglycine, ethylenediamine salt, N-methyl reduced glucosamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine An amine salt of a salt, a tetramethylammonium salt, an organic salt of a hydroxymethylaminomethane salt; a halogen atom, such as a hydrofluoride, a hydrochloride, a hydrobromide or a hydroiodide An acid salt of a nitrate, a perchlorate, a sulfate, a phosphate, or the like; a lower alkanesulfonate such as a methanesulfonate, a trifluoromethanesulfonate or an ethanesulfonate; Acid salt, aryl sulfonate of p-tosylate; acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleic acid An organic acid salt of a salt or the like; and an amine acid salt such as a glycinate, an amidate, a arginine, an alanate, a glutamate or an aspartate. As a more preferable salt, an alkali metal salt such as a sodium salt, a potassium salt or a lithium salt; and, for example, t-octylamine salt, dibenzylamine salt, porphyrin salt, glucosamine salt, phenylglycol Alkylamine salt, ethylenediamine salt, N-methyl reduced glucosamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzyl Ethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine An amine salt of a salt, a tetramethylammonium salt, an organic salt of a hydroxymethylaminomethane salt or the like. Further, preferred examples of the salt include a sodium salt, a potassium salt, a lithium salt, a triethylamine salt, and a hydroxymethylaminomethane salt.

The composite system of the present invention is preferably in the form of a triple helix. In a preferred embodiment, among the three strands forming the triple helix structure, the double strand is a β-1,3-glucan chain, and the single strand is the polydeoxygen gland in the above-mentioned oligodeoxynucleotide of the present invention. Chain of glycosides. Further, the composite may also include a portion that does not form a triple helix structure.

The composition ratio of the oligodeoxynucleotide to the β-1,3-glucan in the complex of the present invention is due to the chain length of the polydeoxyadenosine in the oligodeoxynucleotide, and β-1 The length of 3-glucan can be varied and the like. For example, in the case where the length of the β-1,3-glucan chain and the polydeoxyadenylate chain are equal, the double-stranded β-1,3-glucan chain and the single-stranded invention are The oligodeoxynucleotide combination can form a triple helix structure. In general, since the chain length of polydeoxyadenosine is short relative to the β-1,3-glucan chain, the present invention is plural for the double-stranded β-1,3-glucan chain. The oligodeoxynucleotides are combined by polydeoxyadenosine to form a triple helix structure (see Fig. 10).

The composite system of the present invention contains humanized K-type CpG ODN and β-1,3-glucan (for example, lentinan, schizophyllum polysaccharide, scleroglucan, carterin, lycium polysaccharide, ash tree flower polysaccharide, The complex of laminarin is preferably a complex composed of humanized K-type CpG ODN and β-1,3-glucan (for example, lentinan, schizophyllum polysaccharide, scleroglucan). More preferably, it comprises "on the 3' side of the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, 20 to 60 nucleotides in length (specifically, 20, 21, 22, 23) , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 a polydeoxyadenosine compartment of 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides in length comprising the following formula

a group represented by either of them, or an oligodeoxynucleotide in which all of the phosphodiester bonds are substituted by a phosphorothioate bond, in which the group is repeated 2 or 3 times. And a complex of "β-1,3-glucan (for example, lentinan, Schizophyllum polysaccharide)" (for example, K3-spacer-dA20~60-LNT, K3-spacer-dA20~60-SPG) More preferably, it comprises "on the 3' side of the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, 30 to 50 nucleotides in length (specifically, 30, 31, 32, Polydeoxyadenosine compartments of 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length) Contains the following

a group represented by either of them, or an oligodeoxynucleotide in which all of the phosphodiester bonds are substituted by a phosphorothioate bond, in which the group is repeated 2 or 3 times. And a complex of "β-1,3-glucan (for example, lentinan, Schizophyllum polysaccharide)" (for example, K3-spacer-dA30~50-LNT, K3-spacer-dA30~50-SPG) Further preferably, it comprises "on the 3' side of the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, 30 to 45 nucleotides in length (specifically, 30, 31, 32) a polydeoxyadenosine compartment of 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 nucleotides in length) comprising the formula

a group represented by either of them, or an oligodeoxynucleotide in which all of the phosphodiester bonds are substituted by a phosphorothioate bond, in which the group is repeated 2 or 3 times. And a complex of "β-1,3-glucan (for example, lentinan, Schizophyllum polysaccharide)" (K3-spacer-dA30~45-LNT, K3-spacer-dA30~45-SPG), most Preferably, it comprises "the 3' side of the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, and a 40-nucleotide length polydeoxyadenylate spacer

Any one of the oligodeoxynucleotides in which the base group is repeated 2 or 3 times and the bonded group is bonded, and the phosphodiester bond is replaced by a phosphorothioate bond, and the mushroom Complex of polysaccharide or Schizophyllum polysaccharide (K3-(S18)2-dA40-LNT, K3-(S18)2-dA40-SPG, K3-(S12)2-dA40-LNT, K3-(S12)2 -dA40-SPG).

The preparation of the composite of the present invention can be carried out in the same manner as described in Non-Patent Documents 21 to 24 or JP-A-2008-100919. That is, the β-1,3-glucan originally present in the natural triple helix structure is dissolved in an aprotic organic polar solvent (dimethylammonium (DMSO), acetonitrile, acetone, etc.) or an aqueous alkali solution (hydrogen hydroxide). Sodium, potassium hydroxide, ammonia, calcium hydroxide, etc.) are unwrapped into a single strand. The solution of the single-stranded β-1,3-glucan thus obtained and the solution of the oligodeoxynucleotide of the present invention (aqueous solution, pH-buffered aqueous solution near neutral, or acidic buffered aqueous solution, preferably) Mix with an aqueous solution or a buffered aqueous solution of pH near neutral, and if necessary, adjust the pH to near neutral again and maintain the appropriate time. For example, save at 5 ° C overnight. Double-stranded β-1,3-glucan The chain forms a triple helix structure with the polydeoxyadenosine chain in the oligodeoxynucleotide to form a complex of the invention. The oligodeoxynucleotide in which the complex is not formed can be removed by subjecting the resulting complex to purification by size exclusion chromatography, ultrafiltration, dialysis or the like. Further, by purifying the resulting complex by anion exchange chromatography, the β-1,3-glucan which does not form a complex can be removed. The complex can be appropriately purified by the above method.

The composite of the present invention can be formed by measuring, for example, a configuration change using CD (circular dichroism) spectroscopy, UV absorption displacement using size exclusion chromatography, colloidal electrophoresis, microchip electrophoresis, capillary electrolysis It is confirmed by swimming, but it is not limited to this.

The mixing ratio of the oligodeoxynucleotide of the present invention and β-1,3-glucan can be suitably set in consideration of the length of the polydeoxyadenosine chain, etc., but usually the molar ratio (β-1,3- The dextran (LNT, etc.) / ODN) is 0.02 to 2.0, preferably 0.1 to 0.5. In other aspects, the molar ratio (β-1,3-glucan (LNT, etc.)/ODN) is, for example, 0.005 to 1.0, preferably 0.020 to 0.25.

In another aspect, the mixing ratio of the oligodeoxynucleotide of the present invention and the β-1,3-glucan can be appropriately set in consideration of the length of the polydeoxyadenosine chain, etc., for example, polyoxygenation can be performed. The ratio of the adenosine chain adenosine number (dA) to the β-1,3-glucan backbone glucose number (mG) is set. Usually, the mG/dA ratio is 2.0 to 10, preferably 2.0 to 6.0, more preferably 2.0 to 4.0, still more preferably 2.0 to 3.0.

Regarding the preparation method of the composite of the present invention, a CpG-spacer-ODN and LNT composite will be described as an example. LNT is dissolved in 0.05~2N, preferably 0.1~1.5N alkali aqueous solution (for example, 0.25N sodium hydroxide aqueous solution), and placed at 1 °C ~ 40 °C for 10 hours to 4 days (for example, one night at room temperature) And, a single-stranded LNT aqueous solution (for example, a 50 mg/ml LNT aqueous solution) is prepared. The aqueous solution of LNT and the separately prepared aqueous solution of CpG-spacer-ODN (for example, 100 μM aqueous solution of CpG) are mixed at a molar ratio (LNT/ODN) of 0.005 to 1.0, followed by the aforementioned mixture of LNT and CpG-spacer-ODN. Neutralization is carried out by adding an acidic buffer aqueous solution (for example, NaH ₂ PO ₄ ), and maintaining at 1 to 40 ° C for 6 hours to 4 days (for example, one night at 4 ° C), whereby the complexation is completed. Further, for the above-mentioned compounding, the LNT aqueous solution may be added and mixed at the end. The formation of the complex can be confirmed, for example, by using size exclusion chromatography, and shifting the CpG-spacer-ODN to the high molecular weight side by monitoring the absorption at 240 to 280 nm (for example, 260 nm).

In one aspect, the composite system of the present invention is in the form of a halo-like particle. The particle diameter system is equivalent to the diameter of particles naturally formed by a triple helix structure of β-1,3-glucan (for example, Schizophyllum polysaccharide) used as a material, and usually has an average particle diameter of 10 to 100 nm, preferably It is 20-50 nm. The particle diameter allows the complex to be dissolved or dispersed in water and measured by dynamic light scattering at 80 ° C using a Malvern Instruments Zeta Sizer.

The composite of the present invention is preferably isolated. The purity of the "isolated composite" (the percentage of the weight of the intended composite as a percentage of the total weight of the object to be evaluated) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and further preferably It is over 99%.

The complex of the present invention has excellent immunostimulating activity, in particular, has an immunostimulating activity specific to K-type CpG ODN (for example, B cells (preferably human B cells) are activated to produce IL-6), and D-type CpG ODN-specific immunostimulating activity (for example, plasmacytoid dendritic cells (preferably human plasmacytoid cells) Since dendritic cells are activated to produce IFN-α, both of them are useful as immunostimulating agents. For example, the complex of the present invention comprising an oligodeoxynucleotide (for example, SEQ ID NO: 2 to 17) and LNT (for example, K3-spacer-LNT) and the oligodeoxynucleotide containing the present invention (for example, Sequence identification number 2~17) and SPG complex (eg, K3-spacer-SPG) have inflammatory response-inducing ability (pan-IFN-a, IL-6, etc.), antigen-specific IgG in serum of virus-inoculated individuals Enhancement of antibody valence (total IgG, IgG2c, etc.), antigen-specific interleukin production ability (IFN-γ, IL2, etc.) in virus-inoculated individuals, and anti-viral infection-preventing effect. For K3-spacer-LNT, the virus-inoculated individuals also have the ability to produce Th2-type interleukins (IL-13, etc.). Therefore, there are candidates for use as novel vaccine adjuvants.

3. Pharmaceutical composition

The present invention provides a pharmaceutical composition comprising the above-described oligodeoxynucleotide of the present invention or the above-described complex of the present invention. The pharmaceutical composition of the present invention can be obtained by formulating the above-described oligodeoxynucleotide of the present invention or the above-described complex of the present invention by a usual means. The pharmaceutical composition of the present invention comprises the oligodeoxynucleotide or complex of the present invention and a pharmacologically acceptable carrier. Further, the pharmaceutical composition may further contain an antigen. Such pharmaceutical compositions are provided as a dosage form suitable for oral or parenteral administration.

As a composition for parenteral administration, for example, it can be used for an injection, a suppository, etc., and the injection can include an intravenous injection, a hypodermic injection. A dosage form such as an injection, an intradermal injection, an intramuscular injection, or a drip injection. Such an injection can be prepared in accordance with a known method. The preparation method of the injection can be prepared, for example, by dissolving or suspending the above-described oligodeoxynucleotide or complex of the present invention in a sterile aqueous solvent which is usually used for an injection. As the aqueous solvent for injection, for example, distilled water; physiological saline; a buffer solution such as a phosphate buffer, a carbonate buffer, a Tris buffer, or an acetate buffer can be used. The pH of such an aqueous solvent may be 5 to 10, preferably 6 to 8. The prepared injection is preferably filled in a suitable ampoule.

Further, the suspension of the oligodeoxynucleotide or complex of the present invention may be prepared by vacuum drying, freeze-drying or the like to prepare a powder preparation of the oligodeoxynucleotide or complex of the present invention. The oligodeoxynucleotide or complex of the present invention is stored in a powder state, and the powder is dispersed in an aqueous solvent for injection at the time of use, and can be used for use.

The content of the oligodeoxynucleotide or complex of the present invention in the pharmaceutical composition is usually from about 0.1 to 100% by weight, preferably from about 1 to 99% by weight, and more preferably from about 10% by weight to the total of the pharmaceutical composition. About 90% by weight.

The pharmaceutical composition of the present invention may contain the oligodeoxynucleotide or complex of the present invention as an active ingredient alone, or may be contained by combining the oligodeoxynucleotide or complex of the present invention with other active ingredients.

4. Medical use

Since the oligodeoxynucleotide and the complex of the present invention have excellent immunostimulating activity, the oligodeoxynucleotide, complex and pharmaceutical composition of the present invention can be used as an immunostimulating agent. Oligodeoxynucleosides of the present invention The acid, complex or pharmaceutical composition is administered to a mammal (a primate such as a human or the like, a rodent of a mouse or the like) to induce an immune response in the mammal. In particular, the complex of the present invention has the active property of D-type CpG ODN, which stimulates peripheral blood mononuclear cells, and causes type I interferons (Pan-IFN-α, IFN-α2, etc.) and type II interferons (IFN-γ). Both of them are produced in large quantities, and thus are useful as inducers for type I interferon production, inducers for type II interferon production, and inducers for type I and type II interferon production. The complex of the present invention and the pharmaceutical composition containing the same are useful for preventing or preventing the disease or any of the type I and type II interferons by inducing the production of both type I and type II interferons. treatment. Examples of diseases in which type I interferon is effective include viral infections (for example, hepatitis C virus (HCV), herpes virus, papilloma virus, RS virus, influenza virus, etc.), cancer, and the like. Examples of diseases in which type II interferon is effective include allergic diseases, intracellular parasitic protozoa (leishmania), or bacteria (listeria, tuberculosis, etc.). Etc. For acute viral infections involving RS virus or influenza virus, type I interferon and type II interferon simultaneously increase the immune response associated with virus elimination, so the complex of the present invention and the pharmaceutical composition containing the same can be expected to be acute virus The effectiveness of the infection.

Further, the oligodeoxynucleotide and complex of the present invention, particularly the complex of the present invention, have potent vaccine adjuvant activity, and the oligodeoxynucleotide and complex of the present invention are administered together with an antigen to In mammals, an immune response against the antigen can be strongly induced. Accordingly, the present invention also provides a method for inducing immunity against the antigen comprising "(a) the oligodeoxynucleotide of the present invention, or the complex of the present invention", and "(b) antigen" The composition of the reaction. In particular, the complex of the present invention strongly induces both an anti-antigen humoral immune response (antigen-specific antibody production) and a cellular immune response (antigen-specific CTL induction). Therefore, the oligodeoxynucleotide, complex, and pharmaceutical composition of the present invention, particularly the complex of the present invention and a pharmaceutical composition containing the same, are useful as vaccine adjuvants.

In the present specification, an adjuvant means an auxiliary agent that promotes an immune reaction, and a substance that non-specifically enhances an immune response against the antigen when administered together with an antigen to a living body.

As far as the antigen is concerned, it is antigenic, antibody or cytotoxic T lymphocytes (CTL, CD8 ⁺ T cells) for mammals to be administered (primates such as humans, rodents such as mice, etc.). It can be identified as an antigen, and is not particularly limited, and all substances (proteins, peptides, nucleic acids, lipids, saccharides, and modifications of the above substances) which are antigens can be used (for example, introduction of one or more amino acids) Deletion, substitution, and/or addition, etc. (hereinafter, variants, etc.), etc.) For antigens, antigens derived from pathogens such as protozoa, fungi, bacteria, viruses, etc., and cancer or specific Disease-related antigens.

In the present specification, "antigen A is derived from pathogen X" means that antigen A is contained as a constituent factor in the pathogen X. For example, in the case where the antigen A is a multi-peptide, it means that the amino acid sequence of the multi-peptide is present in the amino acid sequence of the protein encoded by the genome of the pathogen X.

Examples of the antigen derived from the pathogen include a pathogen itself or a part thereof, a pathogen which is not activated or attenuated, or a part thereof, or a modification in which a mutation or the like is introduced.

Inducing resistance when using an antigen from a pathogen as an antigen The immune response of the antigen establishes a mechanism for immunologically excluding pathogens containing the antigen to an ex vivo. Therefore, the composition for inducing an immune response against the antigen is contained in (a) "oligodeoxynucleotide of the present invention, or a complex of the present invention", and (b) "antigen derived from a pathogen" There is a prevention or treatment for a disease caused by the pathogen (for example, an infection of a pathogen).

The complex of the present invention strongly induces both an anti-antigen humoral immune response (antigen-specific antibody production) and a cellular immune response (antigen-specific CTL induction). Therefore, an antigen derived from an intracellular infectious pathogen (virus, protozoa, fungus, bacteria, etc.) known to be recognized by a cytotoxic T lymphocyte, or an antigen associated with a cancerous cell (for example, a tumor antigen) It is suitable to be used as an antigen.

The intracellular infectious virus is not particularly limited, but examples thereof include RS virus, influenza virus, parainfluenza virus, hepatitis C virus (HCV), hepatitis A virus (HAV), and hepatitis B virus (HBV). ), Ebola virus, cytomegalovirus, adenovirus, poliovirus, Japanese encephalitis virus, measles virus, mumps virus, rubella virus, rabies virus, Yellow fever virus, varicella-zoster virus, hantavirus, dengue virus, Norovirus, rotavirus, parvovirus, Coronavirus, distemper virus, adult T cell leukemia virus (HTLV-1), human immunodeficiency virus (HIV), herpes virus, papilloma virus, and the like. Examples of the intracellular infectious bacteria include mycoplasma and the like. Examples of the intracellular infectious protozoa include malaria parasites and schistosomiasis. The intracellular infectious pathogen is preferably a virus (specifically, an RS virus, or an influenza virus, etc.).

Examples of the antigen associated with the cancerous cell include a protein, a sugar chain, a peptide, and a variant (deletion, substitution, and/or addition) of the substance described above, or a modification thereof. Body and so on.

The complex of the present invention strongly induces both type I and type II interferons, and in one aspect, both type I interferons and type II interferons can be selected as effective viruses causing acute viral infections (for example, RS virus, influenza virus) as a virus.

For example, by using (a) "oligodeoxynucleotide of the present invention, or a complex of the present invention", and (b) "antigen derived from a pathogen or cancer", an immune response against the antigen is used. The composition is administered to a human suffering from a pathogen infection or cancer, a human having a possibility of infection with the pathogen or cancer, and the cytotoxic T lymphocyte (CTL) antigen specific to the subject receiving the administration Sexually activating, inducing the production of antibodies specific for the antigen, i.e., by inducing a defense immune response in a warm-blooded animal, preferably a human, can prevent the treatment of the infection or cancer. In other words, the composition is used as a vaccine for the prevention or treatment of diseases such as the above-mentioned infectious diseases and cancer.

Further, the complex of the present invention can strongly induce both a humoral immune response against antigen (antigen-specific antibody production) and a cellular immune response (antigen-specific CTL induction), so that pathogens or cancer cells can also be used. Any of the surface antigen and the internal antigen may be used as an antigen, and it may be expected to mix a surface antigen with an internal antigen.

The composition for inducing an immune response against the antigen (a) "the oligodeoxynucleotide of the present invention or the complex of the present invention", and (b) "antigen" may be in accordance with the pharmaceutical composition of the present invention described above. Object to modulate.

The contents of all publications including the patents and patent application specification incorporated in the specification are hereby incorporated by reference in their entirety in the entireties in

[Examples]

Hereinafter, the present invention will be described in further detail by way of examples, but the invention is not limited to the examples.

[Examples 1-3 and Reference Example 1]

<Synthesis of K3-spacer of the Example and Reference Example K3-dA40>

The K3-spacer of the examples and the reference example K3-dA40 are shown in Table 2, and were synthesized as follows.

In Table 2, the left end indicates the 5' end and the right end indicates the 3' end. The X moiety (also referred to as the spacer moiety) is selected from S18, S12, S9 or C12, and n represents the number of repeats. In the case of n=1, there are cases where it is not specifically described. Is a K3 with a spacer and dA40 (also labeled as K3-spacer), and repeats S18 as a spacer (or two S18 units), labeled K3-(S18)2-dA40.

As, Cs, Gs, Ts, A, S18, S12, S9, and C12 are represented by the following structures.

The oligodeoxynucleotide method uses a solid phase method of the conventional method (Nucleic Acids in Chemistry and Biology, 3. Chemical synthesis (1990) ed. G. Michael Blackburn and Michael J. Gait. Oxford University Press ) to synthesize. When synthesizing part of S18, Spacer 18 (DMT-Hexa-ethoxy-glycol phosphor) was used. Amidite, ChemGene, catalog number: CLP-9765), when using part of S12, use Spacer 12 (DMT-Tetraethoxy-glycol phosphoramidite, ChemGene, catalog) No.: CLP-1368). When synthesizing S9, Spacer 9 (DMT-Triethoxy-glycol phosphoramidite, ChemGene, catalog number: CLP-1113) was used. In the synthesis of C12, Carbon 12 (DMT-Dodecane-Diol phosphoramidite, ChemGene, catalog number: CLP-1114) was used.

Table 3 shows the molecular weight (calculated value) of K3-spacer shown in Table 2, the measured value by mass analysis, and the retention time in the case of analysis by reverse phase HPLC under the following conditions (column: Waters-, X-Bridge C18) 2.5 μm, 4.6 x 75 mm, solution A: 100 mM hexafluoroisopropanol, 8 mM triethylamine, solution B: methanol, B%: 5% → 30% (20 minutes, linear gradient); 60 ° C; 1 ml/min ;260nm).

[Reference Example 2]

<Modulation of K3-dA40 and SPG Complex (K3-SPG Complex)>

[its 1]

7.22 mg of K3-dA40 was dissolved in water (3.7 mL). 15 mg of SPG (Mitsui Sugar) was dissolved in 0.25 N NaOH (1 mL). 1 mL of 330 mM NaH ₂ PO _{4 was} added to the DNA solution, and then the SPG solution was added to the DNA/NaH ₂ PO ₄ solution, and the complexation was completed by allowing to stand overnight at 4 °C. The molar ratio (MSPG/MDNA) was fixed at 0.27. The formation of the complex was confirmed by a microchip electrophoresis apparatus (SHIMADZU: MultiNA).

[its 2]

SPG (Mitsui Sugar) was dissolved in 0.25 N NaOH in a manner of 15 mg/mL, and allowed to stand overnight at room temperature. K3-dA40 was dissolved in water for injection so as to be 100 μM. SPG 8.12 mg was mixed with K3-dA40 4.17 mg, and then 330 mM NaH ₂ PO ₄ in the same volume (541.4 μl) as SPG was added, and the complexation was completed by standing overnight at 4 °C. The formation of the complex was confirmed by size exclusion chromatography, and the shift of K3-dA40 to the high molecular weight side was monitored by monitoring the absorption at 260 nm. The retention time relative to K3-dA40 was 30.35 minutes and the retention time of the K3-SPG complex was 21.99 minutes. Also, since the peak shift was 100%, the formation of the K3-SPG complex was confirmed (system: Agilent 1100 series, column: Asahipak GS-520 HQ (Shodex)-GS-320 HQ (Shodex), flow rate: 0.5 mL /min, buffer: 100 mM phosphate buffer, pH 7.4, temperature: 40 ° C).

[Example 4]

<K3-spacer and LNT complex (K3-spacer-LNT complex) Modulation>

Lentinan (LNT: Ajinomoto Co., Ltd., lot number: 2D8X1) was dissolved in 0.25 N NaOH in a manner of 50 mg/mL, and allowed to stand overnight at room temperature. K3-spacer was dissolved in water for injection so as to be 100 μM. The LNT aqueous solution and the K3-spacer aqueous solution were mixed at a ratio shown in Table 4, and then 330 mM NaH ₂ PO _{4 in the} same volume as the added LNT was added, and the composite was completed by standing at 4 ° C overnight (hereinafter, labeled For K3-spacer-LNT). The formation of the complex was confirmed by size exclusion chromatography, and the displacement of the K3-spacer to the high molecular weight side was monitored by monitoring the absorption at 260 nm.

Analysis condition A

System: Agilent 1100 series, column: Asahipak GF7M-HQ (Shodex) two-tube linkage, flow rate: 0.8 mL/min, buffer: 10 mM EDTA PBS, pH 7.4, temperature: 40 ° C

Analysis condition B

System: Agilent 1100 series, column: Asahipak GS-520 HQ (Shodex)-GS-320 HQ (Shodex), flow rate: 0.5 mL/min, buffer: 100 mM phosphate buffer, pH 7.4, temperature: 40 ° C

Analysis condition C

System: Agilent 1100 series, column: Asahipak GS-520 HQ (Shodex)-GS-320 HQ (Shodex), flow rate: 0.5 mL/min, buffer: 20% acetonitrile, 80 mM phosphate buffer, pH 7.4, temperature :40°C

Retaining time of various K3-spacers and various K3-spacer-LNT complexes in the above analysis conditions, complex K3-spacer-LNT The content (%) of the compound is shown in Table 5. The peak shift of K3-spacer was confirmed in all, so the formation of the K3-spacer-LNT complex was confirmed.

[Example 5]

<Modulation of K3-spacer and SPG Complex (K3-spacer-SPG Complex)>

The Schizophyllum polysaccharide (SPG: Mitsui Sugar Co., Ltd.) was dissolved in 0.25 N NaOH at a rate of 15 mg/mL, and allowed to stand overnight at room temperature. K3-spacer was dissolved in water for injection so as to be 100 μM. The SPG aqueous solution and the K3-spacer aqueous solution were mixed at a ratio shown in Table 6, followed by the addition of 330 mM NaH ₂ PO _{4 in the} same volume as the added SPG, and the complexation was completed by standing overnight at 4 ° C (later, marking For K3-spacer-SPG). The formation of the complex was confirmed by size exclusion chromatography, and the displacement of the K3-spacer toward the high molecular weight side was confirmed by detecting the absorption in 260 nm.

Analysis condition B

System: Agilent 1100 series, column: Asahipak GS-520 HQ (Shodex)-GS-320 HQ (Shodex), flow rate: 0.5 mL/min, buffer: 100 mM phosphate buffer, pH 7.4, temperature: 40 ° C

Analysis condition C

System: Agilent 1100 series, column: Asahipak GS-520 HQ (Shodex)-GS-320 HQ (Shodex), flow rate: 0.5 mL/min, buffer: 20% acetonitrile, 80 mM phosphate buffer, pH 7.4, temperature :40°C

Table 7 shows the retention times of various K3-spacers and various K3-spacer-SPG complexes and the content (%) of the K3-spacer-SPG complex in the above analysis conditions. All of them confirmed the peak shift of K3-spacer at a ratio of more than 90%, so the formation of the K3-spacer-SPG complex was confirmed.

[Test Example 1]

(1) Method

<animal>

C57BL/6J mice were purchased from CLEA, Japan. All animal experiments were carried out in accordance with the guidelines of the Institute of Medicine and the Department of Animals of Osaka University.

<Modulation and stimulation of human PBMCs (Figures 1, 4, 7)>

Frozen human PBMCs (Lonza, Cat# CC-2702, Lot# 0000396517) were thawed and adjusted to 1 x 10 ⁷ cells/ml with complete RPMI (RPMI 1640 supplemented with 10% FCS, penicillin, and streptomycin) density. The cells were seeded in a U-bottom 96-well cell culture dish at 100 μL/well, and stimulated with each vaccine adjuvant for 24 hours. After stimulation, pan-IFN-α (Mabtech) and IL-6 (R&D) in the culture supernatant were quantified by interleukin ELISA. #1 or #2 attached to K3-spacer-LNT in Fig. 1 each refers to an adjuvant complex prepared under the condition that the mG/dA molar ratio is 2.5 or 3.0.

<Anti-RSV F vaccine antigen antibody titer measurement (Figures 2, 5, 8)>

At the base of the tail of 7-week-old C57BL/6 mice, a mixed preparation of 0.5 μg of RSV F antigen and 10 μg of various adjuvants (in a nucleic acid adjuvant, 10 μg of nucleic acid) was inoculated twice at 2 week intervals. One week after the final immunization, recovery of peripheral blood and preparation of serum were performed as evaluation samples. The total IgG and IgG subtypes bound to the RSV F vaccine antigen in serum were determined by ELISA.

<RSV F vaccine antigen-specific T cell interleukin production Ability (Figures 3, 6, and 9) >

At the base of the tail of 7-week-old C57BL/6 mice, 0.5 μg of RSV F antigen and 10 μg of various adjuvants were inoculated twice at 2 week intervals. The spleen was recovered 1 week after the final immunization, and the spleen cells were prepared. The spleen cells inoculated in a 96-well culture plate, each of which is a MHC class I-restricted polypeptide peptide of the RSV F antigen, and a type II MHC binding antigen epitope peptide ( MHC class II-restricted algorithm peptide, vaccine antigen protein to stimulate, culture for 24 hours or 48 hours. The RSV F antigen-specific interleukin producing ability was evaluated by using a culture supernatant as a sample and using an intercellular ELISA method. In this review, IFN-g of Th1 type interleukin, IL-2 produced by self-activated T cells, IL-5 of Th2 type interleukin and IL-18 were evaluated.

(2) Results

<Inflammatory response inducing ability of K3-spacer-LNT and K3-SPG complex using human PBMCs>

For K3-spacer alone (K3-(S18)-dA40, K3-(S18)2-dA40, K3-(S18)3-dA40), and K3-spacer-LNT complex (K3-(S18)-dA40- LNT#1/#2, K3-(S18)2-dA40-LNT#1/#2, K3-(S18)3-dA40-LNT#1/#2), review the inflammatory response inducing ability in human PBMC. The results are shown in Fig. 1. The generation of pan-IFN-a in the K3-spacer alone stimulation was a limit of detection, suggesting that the K3-spacer-LNT complex has high pan-IFN-a inducing ability. Moreover, the pan-IFN-a inducing ability by the K3-spacer-LNT complex was higher than that of the K3-SPG complex. Furthermore, suggesting that it is caused by the K3-spacer-LNT complex The pan-IFN-a inducing ability did not depend on the length of the spacer. On the other hand, in the IL-6-inducing activity, it was observed that the K3-spacer alone, the K3-spacer-LNT complex, and the K3-SPG complex were equivalent.

<RSV F antigen-specific IgG antibody titer in serum of mice vaccinated with RSV F subunit vaccine supplemented with K3-spacer-LNT and K3-SPG complex>

K3-(S18)-dA40, K3-(S18)2-dA40, K3-(S18)3-dA40 K3-spacer alone, K3-spacer-LNT complex (K3-(S18)-dA40-LNT, K3-(S18)2-dA40-LNT, K3-(S18)3-dA40-LNT), and K3-SPG antibody-promoting activity by measuring antibody potency in serum of mice immunized with RSV F subunit vaccine Price to evaluate. The results are shown in Fig. 2. RSV F antigen-specific total IgG induction was enhanced by the addition of adjuvant compared to the RSV F antigen alone vaccination group (F), suggesting that K3-spacer alone (F+K3-(S18)-dA40, F+K3 -(S18)2-dA40, F+K3-(S18)3-dA40), K3-spacer-LNT complex (F+K3-(S18)2-dA40-LNT and F+K3-(S18)3- The auxiliary effects of dA40-LNT) and K3-SPG complex (F+K3-dA40-SPG) and alumium phosphate (F+Alum) are equivalent. In the group of mice inoculated with a Th2 type adjuvant of alum (F+Alum), it was observed that the IgG1 subtype induction ability was higher than that of the RSV F antigen alone vaccination group (F), and K3-spacer-LNT The IgG1 inducing ability equivalent to that of alum phosphate was also observed in the complex. On the other hand, regarding the IgG2c subtype antibody, the immunized group inoculated with the K3-spacer alone, the K3-spacer-LNT complex, and the K3-SPG complex showed a highly enhanced activity as compared with alum. From the above results, it is suggested that the K3-spacer-LNT complex including the (S18), (S18)2, and (S18)3 spacers is the same as the K3-SPG complex. The Th1 type adjuvant activity, on the other hand, also has the same Th2 type immunopotentiating activity as alum.

<RSV F antigen-specific interleukin production ability in mice vaccinated with RSV F subunit vaccine supplemented with K3-spacer-LNT and K3-SPG complex>

K3-spacer alone (K3-(S18)-dA40, K3-(S18)2-dA40, K3-(S18)3-dA40), K3-spacer-LNT complex (K3-(S18)-dA40-LNT , K3-(S18)2-dA40-LNT, K3-(S18)3-dA40-LNT), and the RSV F antigen-specific interleukin production-inducing ability of the K3-SPG complex, measured by RSV F The spleen cells of the mouse immunized with the subunit vaccine were evaluated in response to the interleukin produced by antigen stimulation. The results are shown in Figure 3. RSV F antigen-specific IFN-g production induction and IL-2 production-inducing enhancement compared to K3-spacer alone in K3-spacer-LNT complex (F+K3-(S18)-dA40-LNT, F +K3-(S18)2-dA40-LNT, F+K3-(S18)3-dA40-LNT) is higher, suggesting K3-LNT complex and K3-SPG complex (F+K3-dA40-SPG) The activity is equal. In the alum adjuvant adjuvant group (F+Alum) of the Th2 type adjuvant, the stimulation of either of the IFN-g production lines is below the detection limit. On the other hand, the IL-13 production-enhancing effect was observed to be high in the alum phosphate-inoculated group of the Th2-type adjuvant, and was low in the K3-SPG-vaccinated group in the Th1-type adjuvant. Interestingly, in the K3-spacer-LNT complex (F+K3-(S18)-dA40-LNT, F+K3-(S18)2-dA40-LNT, F+K3-(S18)3-dA40-LNT Compared with the K3-SPG complex vaccination group (F+K3-dA40-SPG) of the Th1 type adjuvant, it showed a high IL-13 production-enhancing effect, and a phosphate alum vaccination group belonging to the Th2 type adjuvant. Equivalent. Thus, suggest In addition to the high Th1 reaction enhancing effect of the K3-SPG complex, the K3-spacer-LNT complex also has a Th2 reaction enhancing ability.

<Inflammatory response inducing ability of K3-spacer-LNT and K3-SPG complex using human PBMCs>

For the K3-spacer-LNT complex (K3-(S18)2-dA30-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)2-dA40 -LNT, K3-(S12)3-dA40-LNT, K3-(C12)2-dA40-LNT, K3-(C12)3-dA40-LNT) and K3-SPG complexes, reviewing inflammatory responses in human PBMC Induction ability. The results are shown in Fig. 4. Suggesting that K3-(S18)2-dA30-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)2-dA40-LNT, K3-(S12) The K3-spacer-LNT complex of 3-dA40-LNT has high pan-IFN-a inducing ability compared to the K3-SPG complex. On the other hand, in the K3-spacer-LNT complex of K3-(C12)2-dA40-LNT and K3-(C12)3-dA40-LNT, the pan-IFN-a inducing ability is below the detection limit. Regarding the amount of IL-6 production, the K3-spacer-LNT complex other than K3-(C12)2-dA40-LNT and K3-(C12)3-dA40-LNT was observed to be equivalent to the K3-SPG complex. Induction activity.

<RSV F antigen-specific IgG antibody titer in serum of mice vaccinated with RSV F subunit vaccine supplemented with K3-spacer-LNT and K3-SPG complex>

K3-spacer-LNT complex (K3-(S18)2-dA30-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)2-dA40 -LNT, K3-(S12)3-dA40-LNT, K3-(C12)2-dA40-LNT, K3-(C12)3-dA40-LNT) and K3-SPG complex antibody production increased Strong activity was evaluated by measuring the antibody titer in the serum of mice immunized with the RSV F subunit vaccine. The results are shown in Figure 5. RSV F antigen-specific total IgG induction was enhanced by addition of an adjuvant compared to the RSV F antigen alone vaccination group (F), suggesting that the K3-spacer-LNT complex (F+K3-(S18)2-dA30 -LNT, F+K3-(S18)2-dA35-LNT, F+K3-(S18)2-dA40-LNT, F+K3-(S12)2-dA40-LNT, F+K3-(S12)3 -dA40-LNT, F+K3-(C12)2-dA40-LNT, F+K3-(C12)3-dA40-LNT) and K3-SPG complex (F+K3-SPG) and phosphate alum (F+ The auxiliary effect of Alum) is the same possibility. In the group of mice inoculated with a Th2 type adjuvant of alum (F+Alum), it was observed that the IgG1 subtype induction ability was higher than that of the RSV F antigen alone vaccination group (F), and the K3-spacer-LNT complex was observed. The IgG1 inducing ability equal to or higher than that of alum phosphate was also observed. On the other hand, in the IgG2c subtype antibody, the immunized group inoculated with the K3-spacer-LNT complex and K3-SPG showed higher results than that of alum. From the above results, it is suggested that K3-(S18)2-dA30-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)2-dA40-LNT, The K3-spacer-LNT complex of K3-(S12)3-dA40-LNT, K3-(C12)2-dA40-LNT and K3-(C12)3-dA40-LNT has the same potential for antibody-enhancing activity.

<RSV F antigen-specific interleukin production ability in mice vaccinated with RSV F subunit vaccine supplemented with K3-spacer-LNT and K3-SPG complex>

K3-LNT complex (K3-(S18)2-dA30-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)2-dA40-LNT , K3-(S12)3-dA40-LNT, K3-(C12)2-dA40-LNT, K3-(C1 2) Induction of RSV F antigen-specific interleukin production by 3-dA40-LNT) and K3-SPG complex by measuring cells produced by antigen stimulation by spleen cells immunized with RSV F subunit vaccine Interleukin to evaluate. The results are shown in Fig. 6. The enhancement effect of RSV F antigen-specific IFN-g production induction and IL-2 production induction was compared with the antigen alone administration group (F) in the K3-spacer-LNT complex (F+K3-(S18)2 -dA30-LNT, F+K3-(S18)2-dA35-LNT, F+K3-(S18)2-dA40-LNT, F+K3-(S12)2-dA40-LNT, F+K3-(S12 ) 3-dA40-LNT, F+K3-(C12)2-dA40-LNT, F+K3-(C12)3-dA40-LNT) and K3-SPG complex (F+K3-dA40-SPG) are high the result of. In the alum adjuvant adjuvant administration group (F+Alum) belonging to the Th2 type adjuvant, the stimulation of any of the IFN-g production systems is below the detection limit. On the other hand, the IL-13 production-enhancing effect was observed to be higher in the alum phosphate-inoculated group of the Th2-type adjuvant than in the antigen-administered group alone, and was low in the K3-SPG-vaccinated group in the Th1-type adjuvant. the result of. Regarding the K3-spacer-LNT complex, it showed a high IL-13 production-enhancing effect compared with the K3-SPG vaccination group (F+K3-dA40-SPG) of the Th1 type adjuvant, and a phosphoric acid belonging to the Th2 type adjuvant. The alum vaccination group is equivalent. Thus, it is suggested that K3-(S18)2-dA30-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)2-dA40-LNT, K3- (S12) K3-spacer-LNT complex of 3-dA40-LNT, K3-(C12)2-dA40-LNT, K3-(C12)3-dA40-LNT has equivalent Th1 and Th2 interleukin production enhancing activity .

<Review of Inflammatory Response Inducing Ability of K3-spacer-LNT and K3-SPG Complex Using Human PBMCs>

For the K3-spacer-LNT complex (K3-(S18)-dA35-LNT, K3-(S18)-dA40-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3-(S12)-dA35-LNT, K3-(S12)-dA40- LNT, K3-(S12)2-dA35-LNT, K3-(S12)2-dA40-LNT, K3-(S9)-dA35-LNT, K3-(S9)-dA40-LNT, K3-(S9)2 -dA35-LNT, K3-(S9)2-dA40-LNT) and K3-SPG complex, reviewing the inflammatory response inducing ability in human PBMC. The results are shown in Figure 7. The entire K3-spacer-LNT complex, compared to the K3-SPG complex, is indicated to have high pan-IFN-a inducing ability. On the other hand, the IL-6 production amount was observed to be equivalent to the induction activity of the K3-spacer-LNT complex and the K3-SPG complex.

<RSV F antigen-specific IgG antibody titer in serum of mice vaccinated with RSV F subunit vaccine supplemented with K3-spacer-LNT complex>

K3-spacer-LNT complex (K3-(S18)-dA35-LNT, K3-(S18)-dA40-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT , K3-(S12)-dA35-LNT, K3-(S12)-dA40-LNT, K3-(S12)2-dA35-LNT, K3-(S12)2-dA40-LNT, K3-(S9)-dA35 -LNT, K3-(S9)-dA40-LNT, K3-(S9)2-dA35-LNT, K3-(S9)2-dA40-LNT) antibodies produce potentiating activity by measuring RSV F subunit vaccine The antibody titer in the serum of the immunized mice was evaluated. The results are shown in Fig. 8. The RSV F antigen-specific total IgG induction was enhanced by the addition of an adjuvant compared to the RSV F antigen alone vaccination group (F), suggesting that the K3-spacer-LNT complex (F+K3-(S18)-dA35- LNT, F+K3-(S18)-dA40-LNT, F+K3-(S18)2-dA35-LNT, F+K3-(S18)2-dA40-LNT, F+K3-(S12)-dA35- LNT, F+K3-(S12)-dA40-LNT , F+K3-(S12)2-dA35-LNT, F+K3-(S12)2-dA40-LNT, F+K3-(S9)-dA35-LNT, F+K3-(S9)-dA40-LNT The adjuvant effect of F+K3-(S9)2-dA35-LNT, F+K3-(S9)2-dA40-LNT) and alumole phosphate (F+Alum) is equivalent. In the mouse group inoculated with a Th2 type adjuvant of alum (F+Alum), it was observed that the IgG1 subtype inducing ability was higher than that of the RSV F antigen alone vaccination group (F), and the longest spacer length was K3. -(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT complex administration group (F+K3-(S18)2-dA35-LNT, F+K3-(S18)2-dA40- LNT) was also observed to have the same IgG1 inducing ability as alum. On the other hand, in the IgG2c subtype antibody, the immunized group inoculated with the whole K3-spacer-LNT complex showed higher results than the alum. From the above results, it is suggested that K3-(S18)-dA35-LNT, K3-(S18)-dA40-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT, K3- (S12)-dA35-LNT, K3-(S12)-dA40-LNT, K3-(S12)2-dA35-LNT, K3-(S12)2-dA40-LNT, K3-(S9)-dA35-LNT, The K3-spacer-LNT complex of K3-(S9)-dA40-LNT, K3-(S9)2-dA35-LNT, K3-(S9)2-dA40-LNT has the same Th1 enhancing activity, especially K3 -(S18)2-dA35-LNT and K3-(S18)2-dA40-LNT have high Th2 enhancing activity.

<RSV F antigen-specific interleukin production ability in mice vaccinated with RSV F subunit vaccine supplemented with K3-spacer-LNT complex>

K3-spacer-LNT complex (K3-(S18)-dA35-LNT, K3-(S18)-dA40-LNT, K3-(S18)2-dA35-LNT, K3-(S18)2-dA40-LNT , K3-(S12)-dA35-LNT, K3-(S12)-dA40-LNT, K3- (S12) 2-dA35-LNT, K3-(S12)2-dA40-LNT, K3-(S9)-dA35-LNT, K3-(S9)-dA40-LNT, K3-(S9)2-dA35-LNT , K3-(S9)2-dA40-LNT) The RSV F antigen-specific interleukin production-inducing ability by measuring the interleukin produced by the spleen cells of the mouse immunized with the RSV F subunit vaccine in response to antigen stimulation To evaluate. The results are shown in Figure 9. The enhancement effect of RSV F antigen-specific IFN-g production induction and IL-2 production induction was compared with the antigen alone administration group (F) in the K3-spacer-LNT complex (F+K3-(S18)-dA35 -LNT, F+K3-(S18)-dA40-LNT, F+K3-(S18)2-dA35-LNT, F+K3-(S18)2-dA40-LNT, F+K3-(S12)-dA35 -LNT, F+K3-(S12)-dA40-LNT, F+K3-(S12)2-dA35-LNT, F+K3-(S12)2-dA40-LNT, F+K3-(S9)-dA35 -LNT, F+K3-(S9)-dA40-LNT, F+K3-(S9)2-dA35-LNT, F+K3-(S9)2-dA40-LNT) were high results. In the alum adjuvant adjuvant administration group (F+Alum) which is a Th2 type adjuvant, the stimulation of any of the IFN-g production systems is below the detection limit. On the other hand, the enhancement effect of IL-5 and IL-13 production was higher than that of the antigen alone administration group (F), and the alum phosphate inoculation group of the Th2 type adjuvant was observed to be higher. In the K3-spacer-LNT complex, the K3-(S18)2-dA40-LNT administration group (F+K3-(S18)2-dA40-LNT) showed high IL-5 and IL-13 production enhancing effects. From the above results, it is suggested that the K3-spacer-LNT complex has the same natural immunostimulating ability and Th1 enhancing activity as the K3-SPG complex, and also has a high Th2 in K3-(S18)2-dA40-LNT. Enhance activity.

[Test Example 2]

The following evaluation was performed in the same manner as in Test Example 1.

<Inflammation of the K3-spacer-SPG complex using human PBMCs Review of inducing ability >

For the K3-spacer-SPG complex (K3-(S9)-dA40-SPG, K3-(S9)2-dA40-SPG, K3-(S12)-dA40-SPG, K3-(S12)2-dA40-SPG , K3-(S18)-dA40-SPG, K3-(S18)2-dA40-SPG, K3-(C12)2-dA40-SPG, K3-(C12)3-dA40-SPG), reviewed in human PBMC Inflammatory response inducing ability. The results are shown in Fig. 11. K3-(S9)-dA40-SPG, K3-(S9)2-dA40-SPG, K3-(S12)-dA40-SPG, K3-(S12)2-dA40-SPG, K3-(S18)-dA40 The -SPG, K3-(S18)2-dA40-SPG complex is suggested to have pan-IFN-a, IL-6 inducing ability. On the other hand, in the K3-(C12)2-dA40-SPG, K3-(C12)3-dA40-SPG complex, the inducing activity of pan-IFN-a and IL-6 was the limit of detection.

<RSV F antigen-specific IgG antibody titer in serum of mice vaccinated with RSV F subunit vaccine supplemented with K3-spacer-SPG complex>

K3-spacer-SPG complex (K3-(S9)-dA40-SPG, K3-(S9)2-dA40-SPG, K3-(S12)-dA40-SPG, K3-(S12)2-dA40-SPG , K3-(S18)-dA40-SPG, K3-(S18)2-dA40-SPG, K3-(C12)2-dA40-SPG, K3-(C12)3-dA40-SPG) antibodies produce potentiating activity, Evaluated by measuring the antibody titer in the serum of mice immunized with the RSV F subunit vaccine. The results are shown in Fig. 12. It is clear that the RSV F antigen-specific antibody is induced in the K3-spacer-SPG complex administration group (F+K3-(S9)-dA40-SPG, F+K3 compared to the RSV F antigen alone vaccination group (F). -(S9)2-dA40-SPG, F+K3-(S12)-dA40-SPG, F+K3-(S12)2-dA40-SPG, F+K3-(S18)-dA40-SPG, F+K3 -(S18) It was enhanced in 2-dA40-SPG, F+K3-(C12)2-dA40-SPG, F+K3-(C12)3-dA40-SPG).

<RSV F antigen-specific interleukin production ability in mice vaccinated with RSV F subunit vaccine supplemented with K3-spacer-SPG complex>

K3-spacer-SPG complex (K3-(S9)-dA40-SPG, K3-(S9)2-dA40-SPG, K3-(S12)-dA40-SPG, K3-(S12)2-dA40-SPG , RS3-F antigen specificity of K3-(S18)-dA40-SPG, K3-(S18)2-dA40-SPG, K3-(C12)2-dA40-SPG, K3-(C12)3-dA40-SPG) The interleukin production inducing ability was evaluated by measuring the interleukins produced by the spleen cells of the mice immunized with the RSV F subunit vaccine in response to antigen stimulation. The results are shown in Fig. 13. RSV F antigen-specific IFN-g production induces an enhanced effect on IL-2 production induction, compared to the antigen alone administration group (F), in the K3-spacer-SPG complex (F+K3-(S9)- dA40-SPG, F+K3-(S9)2-dA40-SPG, F+K3-(S12)-dA40-SPG, F+K3-(S12)2-dA40-SPG, F+K3-(S18)- dA40-SPG, F+K3-(S18)2-dA40-SPG, F+K3-(C12)2-dA40-SPG, F+K3-(C12)3-dA40-SPG) were high results. On the other hand, IL-5 is equivalent to the induction level of the antigen alone (F), but the IL-13 production enhancement effect is compared with the antigen alone administration group (F) at K3-spacer- SPG complex (F+K3-(S9)-dA40-SPG, F+K3-(S9)2-dA40-SPG, F+K3-(S12)-dA40-SPG, F+K3-(S12)2- dA40-SPG, F+K3-(S18)-dA40-SPG, F+K3-(S18)2-dA40-SPG, F+K3-(C12)2-dA40-SPG, F+K3-(C12)3 -dA40-SPG) becomes a high result (Fig. 13).

[Industrial availability]

According to the present invention, an oligodeoxynucleotide having excellent immunostimulating activity and a complex containing the same are provided. In particular, the complex of the present invention has both an immunostimulating activity specific to K-type CpG ODN and an immunostimulating activity specific to D-type CpG ODN. In addition, K3-spacer-SPG and K3-spacer-LNT have potent vaccine adjuvant activity, and when the K3-spacer-SPG or K3-spacer-LNT is vaccinated with the antigen, it stimulates the antigen-specific humoral immunity. And both cellular immunity. Therefore, the complex of the present invention is useful as an immunostimulant or vaccine adjuvant in the field of medicine.

The present application is based on Japanese Patent Application No. 2015-057861 (filed on March 20, 2015) filed in Japan, the content of which is entirely incorporated herein.

<110> National Research and Development Corporation Medical Base. health. Nutrition Institute (NATIONAL INSTITUTES OF BIOMEDICAL INNOVATION, HEALTH AND NUTRITION)

<120> CpG-spacer-oligonucleotide complex having immunostimulating activity and use thereof

<130> 092419

<150> JP2015-057861

<151> 2015-03-20

<160> 17

<170> PatentIn version 3.5

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Claims (45)

An oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxynucleotide of polydeoxyadenosine, wherein the polydeoxyadenosine is isolated The spacer is bound to the 3' side of the humanized K-type CpG oligodeoxynucleotide. An oligodeoxynucleotide according to claim 1, wherein the humanized K-type CpG oligodeoxynucleotide is 10 nucleotides or more in length, and comprises 1 or a plurality of unmethylated CpG motifs. . An oligodeoxynucleotide according to claim 1 or 2, wherein the humanized K-type CpG oligodeoxynucleotide comprises the sequence TCGA or TCGT. The oligodeoxynucleotide according to any one of claims 1 to 3, wherein the humanized K-type CpG oligodeoxynucleotide comprises the nucleotide sequence represented by SEQ ID NO: 1. The oligodeoxynucleotide of any one of claims 1 to 4, wherein a part or all of the phosphodiester bond of the oligodeoxynucleotide is substituted with a phosphorothioate bond. The oligodeoxynucleotide of claim 5, wherein all of the phosphodiester bonds of the oligodeoxynucleotide are substituted with a phosphorothioate linkage. The oligodeoxynucleotide of any one of claims 1 to 6, wherein the polydeoxyadenosine is 20 to 60 nucleotides in length. The oligodeoxynucleotide according to any one of claims 1 to 7, wherein the spacer is a group represented by the formula (A) or a group represented by the formula (B), (wherein m is 2, 3, 4, 5, 6, 7, 8, or 9, n is 1, 2, 3, 4, 5 or 6, and formula (3m+2)n is 8 or more and 60 or less, plural R ¹ , R ² , R ³ and R ⁴ are each independently a hydrogen atom or a C _1-6 alkyl group, and X is an oxygen atom or a sulfur atom), (where s is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, t is 1, 2, 3, 4, 5 or 6, and (s+3)t is 8 Above 60, the plural R ⁵ and R ⁶ are each independently a hydrogen atom or a C _1-6 alkyl group, and X is an oxygen atom or a sulfur atom). The oligodeoxynucleotide according to any one of claims 1 to 8, wherein the spacer is a group represented by the formula (A1) or a group represented by the formula (B1), (wherein m is 4, 5 or 6, n is 1 or 2, and the formula (3m+2)n is 14 or more and 40 or less, and the plural R ¹ , R ² , R ³ and R ^{4 are} all a hydrogen atom, X Is a sulfur atom), (wherein s is 8, 9, 10, 11 or 12, t is 1 or 2, (s+3)t is 14 or more and 40 or less, and plural R ⁵ and R ^{6 are} all hydrogen atoms, and X is a sulfur atom. ). The oligodeoxynucleotide according to any one of claims 1 to 9, wherein the spacer is a group represented by any one of the following formulas or a group in which the group is repeated 2 or 3 times, A complex comprising the oligodeoxynucleotide of any one of claims 1 to 10 and β-1,3-glucan. The complex of claim 11, wherein the β-1,3-glucan is lenthinan, schizophyllan, scleroglucan, curdlan, scorpion polysaccharide (pachyman), grifolan, or laminaran. The complex of claim 12, wherein the β-1,3-glucan is lentinan, Schizophyllum polysaccharide or scleroglucan. The complex of claim 11, which comprises the oligodeoxynucleotide described in (i) below, and the β-1,3-glucan described in (ii): (i) comprising the sequence identification number 1 The 3' side spacer of the oligodeoxynucleotide of the indicated nucleotide sequence binds to a polydeoxyadenosine of 20 to 60 nucleotides in length, and the entire phosphodiester bond is phosphorothioate. An ester bond-substituted oligodeoxynucleotide; (ii) a lentinan or a Schizophyllum polysaccharide. The complex of claim 11, which comprises an oligodeoxynucleotide, and a β-1,3-glucan, wherein the oligodeoxynucleotide is contained in the nucleotide sequence represented by SEQ ID NO: 1. Oligodeoxynucleotide 3' side spacer spacer and 20 to 60 nucleotides length of polydeoxyadenylate, and all of the phosphodiester bond substituted by phosphorothioate linkage oligodeoxygenation a nucleotide comprising a group represented by any one of the following formulas or a group bonded by repeating the group 2 or 3 times, The complex of claim 15, which comprises an oligodeoxynucleotide, and a β-1,3-glucan, wherein the oligodeoxynucleotide is contained in the nucleotide sequence represented by SEQ ID NO: 1. Oligodeoxynucleotide 3' side spacer spacer and 30 to 50 nucleotides of polydeoxyadenylate, and all of the phosphodiester bonds are replaced by phosphorothioate linkages a nucleotide comprising a group represented by any one of the following formulas or a group bonded by repeating the group 2 or 3 times, The complex of claim 16, which comprises an oligodeoxynucleotide, and a β-1,3-glucan, wherein the oligodeoxynucleotide is contained in the nucleotide sequence represented by SEQ ID NO: 1. a 3'-side spacer of an oligodeoxynucleotide that binds to a polydeoxyadenosine of 30 to 45 nucleotides in length, and a oligodeoxygen group in which all of the phosphodiester bonds are substituted with a phosphorothioate bond a nucleotide comprising a group represented by any one of the following formulas or a group bonded by repeating the group 2 or 3 times, The complex of claim 17, which comprises an oligodeoxynucleotide, and a lentinan or a Schizophyllum polysaccharide, wherein the oligodeoxynucleotide is oligode selected from the nucleotide sequence represented by SEQ ID NO: 1. a 3'-side spacer of an oxynucleotide that binds to a polydeoxynucleotide of 40 nucleotides in length and a phosphorothioate-substituted oligodeoxynucleotide, The spacer includes a group in which a group represented by any one of the following formulas is repeated 2 or 3 times, and is bonded. The composite of any one of claims 11 to 18, which is a triple helix configuration. The complex according to any one of claims 11 to 19, which has an activity of activating B cells to produce IL-6, and a dendritic cell (dendritic) Cell) The activity which is activated to produce IFN-α. A pharmaceutical composition comprising the oligodeoxynucleotide according to any one of claims 1 to 10, or the complex according to any one of claims 11 to 20. The pharmaceutical composition according to claim 21, which is for use in the prevention or treatment of viral infections, cancer, allergic diseases, intracellular parasitic protozoa or bacterial infections. The pharmaceutical composition of claim 22, which is for use in the prevention or treatment of a viral infection. The pharmaceutical composition of claim 23, wherein the viral infection is an RS virus or an influenza virus infection. A type I and/or type II interferon production inducing agent comprising the complex of any one of claims 11 to 20. An immunostimulant comprising the oligodeoxynucleotide according to any one of claims 1 to 10, or the complex according to any one of claims 11 to 20. An immunostimulant as claimed in claim 26 which is a vaccine adjuvant. A prophylactic or therapeutic agent for a viral infection, a cancer, an allergic disease, an intracellular parasitic protozoan or a bacterial infection, comprising the oligodeoxynucleotide according to any one of claims 1 to 10, or as requested The complex of any one of items 11 to 20. The prophylactic or therapeutic agent according to claim 28, wherein the viral infection is an RS virus or an influenza virus infection. The use of an oligodeoxynucleotide according to any one of claims 1 to 10, or a complex according to any one of claims 11 to 20, for use in the manufacture of a pharmaceutical composition. The use of claim 30, wherein the pharmaceutical composition is a viral infection, A pharmaceutical composition for the prevention or treatment of cancer, allergic diseases, intracellular parasitic protozoa or bacterial infections. The use of claim 31, wherein the viral infection is an RS virus or an influenza virus infection. A method for the treatment or prevention of a disease in a warm-blooded animal, comprising the oligodeoxynucleotide according to any one of claims 1 to 10, or the complex according to any one of claims 11 to 20 The pharmacologically effective amount is administered to the warm-blooded animal. The method of claim 33, wherein the disease is a viral infection, a cancer, an allergic disease, an intracellular parasitic protozoa or a bacterial infection. The method of claim 34, wherein the viral infection is an RS virus or an influenza virus infection. The method of any one of claims 33 to 35, wherein the warm-blooded animal is a human. A method of inducing a defensive immune response in a warm-blooded animal, comprising the pharmacological agent of the oligodeoxynucleotide according to any one of claims 1 to 10, or the complex according to any one of claims 11 to 20 An effective amount is administered to the warm-blooded animal. The oligodeoxynucleotide according to any one of claims 1 to 10, or the complex according to any one of claims 11 to 20, which is used for viral infection, cancer, allergic disease, intracellular Treatment or prevention of parasitic protozoal or bacterial infections. An oligodeoxynucleotide or complex according to claim 38, wherein the viral infection is an RS virus or an influenza virus infection. A pharmaceutical composition comprising (a) according to any one of claims 1 to 10 An oligodeoxynucleotide, or a complex according to any one of claims 11 to 20, and (b) an antigen. The composition of claim 40 is for use in inducing an immune response against the antigen. The composition of claim 41, wherein the antigen is an antigen from a pathogen. The composition of claim 42, which is for the prevention or treatment of an infectious agent of a pathogen. The composition of claim 43, wherein the pathogen is a virus. The composition of claim 44, wherein the virus is an RS virus or an influenza virus.