Compound, immunoadjuvant, vaccine composition and mucosal vaccine composition

Abstract

To provide a compound that exhibits high migration ability to immune cells, enabling more efficient activation of the immune cells.SOLUTION: A compound has a plurality of first ligand moieties binding to C-type lectin receptors, a second ligand moiety binding to Toll-like receptors, and a linker with a branched structure where the first ligand moieties and the second ligand moiety are respectively bound at the terminals.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to compounds, immune enhancing agents, vaccine compositions and mucosal vaccine compositions. [Background technology]

[0002] In the development of vaccines and cancer immunotherapy drugs against emerging or re-emerging infectious diseases, drugs that enhance or regulate the immune system are required. The immune system consists of innate immunity and adaptive immunity. Activation of innate immunity is the starting point of the initial response. Innate immunity is activated when innate immune receptors (also called pattern recognition receptors) recognize specific molecular patterns. Activation of innate immunity is important in the overall immune response of the body because it induces adaptive immunity such as cellular immunity and humoral immunity. Innate immune receptors are expressed on the cell surface and endosomes of immune cells. Ligands of various innate immune receptors that activate innate immunity can be used as immunoregulatory drugs. Innate immune receptor ligands can also be added to vaccine formulations as adjuvants to enhance the effectiveness of immune responses.

[0003] Conventionally, sugars, lipids, proteins, nucleic acids, etc. have been used as ligands for innate immune receptors based on the structures of pathogen-related molecules. For example, mucosal vaccines have been developed that use glycolipids (monophosphoryl lipid A: MPLA, see Non-Patent Document 1) or nucleic acids (PolyI:C, CpG ODN, etc., see Non-Patent Document 2) as adjuvants that can activate the Toll-like receptor (TLR), which is one of the innate immune receptors.

[0004] Mucosal vaccines, which administer antigens to the mucosa, are expected to be the next-generation vaccines because they can be administered non-invasively and easily, and can induce not only local immune responses in the mucosa but also systemic immune responses (see Non-Patent Document 3). For example, sugars and lipids with thickening properties, toxic proteins such as cholera toxin, and inorganic salts such as aluminum salts have been used as adjuvants for mucosal vaccines. These diffuse and are taken up by immune cells in mucosal tissues to induce immune responses, but do not have the ability to activate the innate immune receptors of immune cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Rhea N. Coler, et al., Development and Characterization of Synthetic Glucopyranosyl Lipid Adjuvant System as a Vaccine Adjuvant, PLoS One 6 (2011) e16333. [Non-Patent Document 2] Neslihan Kayraklioglu et al., CpG Oligonucleotides as Vaccine Adjuvants, Methods in Molecular Biology 2197 (2021) 51-85 [Non-Patent Document 3] Marian R. Neutra and Pamela A. Kozlowski, Mucosal vaccines: the promise and the challenge, Nat. Rev. Immunol., 6, (2006) 148-158 [Non-Patent Document 4] Akihito Baba, et al., Synthesis and immunostimulatory activity of sugar-conjugated TLR7 ligands, Bioorganic & Medicinal Chemistry Letters 30 (2020) 126840 [Non-Patent Document 5] RL Miller, et al., Imiquimod applied topically: a novel immune response modifier and new class of drug, Int. J. Immunopharmacol. 21 (1999) 1-14 Summary of the Invention [Problem to be solved by the invention]

[0006] However, sugars, lipids, proteins, and nucleic acids are difficult to formulate because of their heterogeneity, and have low transferability to immune cells. In contrast, Non-Patent Document 4 reports that conjugating 1V209, a hydrophobic TLR7 ligand, to sugar increases hydrophilicity and increases the uptake of 1V209 into cells, thereby enhancing cytokine secretion in immune cells such as dendritic cells compared to cells treated with 1V209 not conjugated to sugar.

[0007] On the other hand, drugs that can activate innate immune receptors and are used in clinical applications include MPLA and imiquimod (see Non-Patent Document 5). MPLA is a lipid-soluble emulsion formulation, and therefore has problems with the stability of the formulation. In addition, imiquimod is limited to local administration, and its range of application is very limited. In addition, drugs that are widely used as vaccine adjuvants include alum and squalene, but these hardly activate innate immune receptors.

[0008] In addition, in the development of mucosal vaccines, it is essential to add an adjuvant that efficiently transports antigens to immune cells present under the mucosal tissue and activates the immune cells. Adjuvants that have both of these functions can be used as universal adjuvants, so they have been actively researched, but have not yet been put to practical use.

[0009] The present invention has been made in view of the above circumstances, and aims to provide a compound and an immune enhancer that have high translocation ability to immune cells and can more effectively activate immune cells. Another aim is to provide a vaccine composition that can efficiently transport an antigen to immune cells and activate the immune cells. Another aim is to provide an immune enhancer and a mucosal vaccine composition that can efficiently transport an antigen to immune cells present under mucosal tissue and activate the immune cells. [Means for solving the problem]

[0010] (compound) The compounds described in this invention are a plurality of first ligand moieties that bind to a C-type lectin receptor; a second ligand portion that binds to a Toll-like receptor; a linker having a branched structure in which the plurality of first ligand moieties and the second ligand moiety are each bonded to an end thereof; has.

[0011] The compound described in the present invention is Formula (1) [ka] (In formula (1), R C has the first ligand moiety, and R T has the second ligand moiety, and p 1 ~p 3 are each independently an integer between 0 and 6.) The semiconductor device may have a structure shown in FIG.

[0012] The structure of the first ligand portion is A structure in which a monosaccharide selected from the group consisting of glucose, galactose, mannose, amino sugars and acidic sugars is bound to the linker; or The structure may be such that one or more oligosaccharides or polysaccharides selected from the group consisting of glucose, galactose, mannose, amino sugars and acidic sugars are bound to the linker.

[0013] The second ligand moiety is It may bind to at least one selected from the group consisting of Toll-like receptor 4, Toll-like receptor 7 and Toll-like receptor 8.

[0014] The structure of the second ligand portion is The structure may be such that 1V209 is bound to the linker.

[0015] (Immuno-boosting agent) The immune enhancing agent described in the present invention is a plurality of first ligand moieties that bind to a C-type lectin receptor; a second ligand portion that binds to a Toll-like receptor; a linker having a branched structure in which the plurality of first ligand moieties and the second ligand moiety are each bonded to an end thereof; The compounds include those having the formula:

[0016] The immune enhancing agent according to the present invention comprises: It may also be an adjuvant for a vaccine.

[0017] The immune enhancing agent according to the present invention comprises: It may be a mucosal immune enhancer.

[0018] The immune enhancing agent according to the present invention comprises: It may be an adjuvant for a mucosal vaccine.

[0019] (Vaccine composition) The vaccine composition according to the present invention comprises: The immune enhancing agent according to the present invention; An antigen; Includes.

[0020] (Mucosal vaccine composition) The mucosal vaccine composition according to the present invention comprises: The immune enhancing agent according to the present invention; An antigen; Includes.

[0021] The mucosal vaccine composition according to the present invention comprises: It may be for administration to the nasal mucosa. Effect of the Invention

[0022] The compound and immune enhancing agent according to the present invention have high translocation ability to immune cells and can more effectively activate immune cells. The vaccine composition according to the present invention can efficiently transport antigens to immune cells and activate immune cells. The immune enhancing agent and mucosal vaccine composition according to the present invention can efficiently transport antigens to immune cells present under mucosal tissue and activate immune cells. [Brief description of the drawings]

[0023] [Figure 1] FIG. 2 is a graph showing the amount of IL-6 produced relative to the concentration of Toll-like receptor 7 ligand substantially added to the culture medium in the treatment of mouse bone marrow-derived dendritic cells with a sugar chain-TLR ligand complex in Test Example 1. [Diagram 2] This is a diagram showing the immune enhancing activity of a sugar chain TLR ligand complex against ovalbumin (OVA) in Test Example 2. (A) and (B) show the IgA antibody production titers against OVA in nasal washings and serum, respectively. (C) and (D) show the IgG antibody production titers against OVA in nasal washings and serum, respectively. [Diagram 3] FIG. 1 shows the spleen weights of mice in Test Example 2. [Figure 4]This is a diagram showing the immune enhancing activity of the glycan-TLR ligand complex against influenza virus-derived hemagglutinin (HA) in Test Example 3. (A) and (B) show the IgA antibody production titers against HA in nasal washings and serum, respectively. (C) and (D) show the IgG antibody production titers against HA in nasal washings and serum, respectively. [Diagram 5] This is a diagram showing the immune enhancing activity of the sugar chain TLR ligand complex against influenza vaccine HA in Test Example 4. (A) and (B) show the IgA antibody production titers against HA in nasal washings and serum, respectively. (C) and (D) show the IgG antibody production titers against HA in nasal washings and serum, respectively. [Figure 6] FIG. 13 shows the results of evaluating the virus neutralizing activity of antibodies produced in the nasal mucosa in Test Example 5. [Figure 7] 1 shows the results of a test for protection against influenza virus infection using mice in Test Example 6. (A) and (B) show the change in body weight over time and the survival rate of mice after infection, respectively. [Figure 8] 1 shows the results of a test for protection against influenza virus infection using mice in Test Example 7. (A) and (B) show the change in body weight over time and the survival rate of mice after infection, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. In each drawing, the same or equivalent parts are given the same reference numerals. Note that the present invention is not limited to the following embodiment and drawings. Note that in the following embodiment, the expressions "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of".

[0025] The immune enhancing agent according to the present embodiment includes a compound. The compound has a CLR ligand portion as a first ligand portion that binds to a C-type lectin receptor (hereinafter also referred to as "CLR"), and a TLR ligand portion as a second ligand portion that binds to a TLR. In the molecular structure, the TLR ligand portion is linked to the CLR ligand portion via a linker. The CLR ligand portion and the TLR ligand portion are linked to each other via a linker means that at least one atom constituting the CLR ligand and at least one atom constituting the TLR ligand are each bonded to or substituted with an atom constituting the linker. The CLR ligand portion refers to an atomic group consisting of the remaining atoms that are not bonded to the linker among the atoms constituting the CLR ligand. Similarly, the TLR ligand portion refers to an atomic group consisting of the remaining atoms that are not bonded to the linker among the atoms constituting the TLR ligand.

[0026] For example, the structure of the above compound is shown in formula (2) below. L C -LL T Formula (2) In formula (2), L C represents a CLR ligand moiety. L represents a linker. T indicates the TLR ligand moiety.

[0027] Preferably, the compound has multiple CLR ligand moieties per molecule. The multiple CLR ligand moieties may all be the same, or may be CLR ligand moieties with different structures. When the compound has multiple CLR ligand moieties per molecule, for example, the multiple CLR ligand moieties may be L C1 and L C2 Then, the structure of the compound is shown in the following formula (3). L C1 -L(-L C2 )-L T Formula (3) In formula (3), L represents a linker. T represents a TLR ligand moiety. In formula (2) and formula (3), L C , LC1 and L C2 Each of the formulas (2) and (3) has a structure in which at least one atom constituting the CLR ligand is replaced with an atom constituting the linker. T is a structure in which at least one atom constituting the TLR ligand is substituted with an atom constituting the linker. In other words, the CLR ligand portion and the TLR ligand portion are included in the structure of the compound according to the present embodiment as a structure derived from the CLR ligand and a structure derived from the TLR ligand, respectively, by bonding with the linker.

[0028] The CLR ligand is not particularly limited as long as it is a molecule capable of binding to the CLR. To date, 18 types of CLR groups having C-type lectin-like domains have been found. Each CLR is thought to recognize a pathogen or an endogenous glycan structure. Examples of CLR ligands include monosaccharides and glycans.

[0029] A monosaccharide is the smallest unit of sugar that cannot be further hydrolyzed. Examples of monosaccharides or sugar chains include glucose, galactose, mannose, fucose, maltose, arabinose, xylose, rhamnose, isomaltose, idose, lactose, panose, cellobiose, melibiose, arabinobiose, isoprimeverose, gentiobiose, mannooligosaccharide chains, chitooligosaccharide chains, laminarioligosaccharide chains, glucosamine, N-acetylglucosamine, glucuronic acid, cyclodextrin, aminocyclodextrin, amylopectin, amylose, arabinan, arabinoxylan, curdlan, dextran, glucomannan, galactomannan, mannan, pullulan, zymosan, xylan, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, hyaluronic acid, alginic acid, carrageenan, fucoidan, polyguluronic acid, polymannuronic acid, chitosan, and chitin. As the monosaccharide, a sugar having sialic acid such as sialyllactose, and a sulfated sugar (a sugar having a sulfate group) such as dextran sulfate may be used. Preferably, the monosaccharide is selected from the group consisting of glucose, galactose, mannose, amino sugars, and acidic sugars.

[0030] The sugar chain is not particularly limited in other configurations as long as it has a plurality of monosaccharides. The sugar chain may be a compound in which a plurality of the above monosaccharides are bound by glycosidic bonds, such as oligosaccharides and polysaccharides. The oligosaccharides and polysaccharides may be cyclic or may have a branched structure (branched structure).

[0031] When the sugar chain is an oligosaccharide or polysaccharide, it may be a single oligosaccharide or polysaccharide consisting of the same monosaccharide, or a complex sugar consisting of various monosaccharides or their derivatives. The monosaccharides constituting the sugar chain may be any of various natural sugars obtained by isolation and purification from nature, or may be artificially synthesized sugars. The above monosaccharides and oligosaccharides may be obtained by decomposing polysaccharides.

[0032] Individuals are Glc, GlcNAc, Gal, Ara, Xyl, Rha, Glcα1-4Glc, and Glcα1-4Glcα 1-4Glc, Glcα1-6Glc, Glcα1-6Glcα1-6Glc, Araα1-5Ara, Glcβ1-3Glcβ1-3 Glc、Glcβ1-4Glc、Glcβ1-6Glc、Galα1-6Glc、Galα1-4Galβ1-4Glc、Galβ1- 3GalNAcα1-6Glc, Galβ1-4GlcNAcβ1-6Glc, Galβ1-4Glc, Galβ1-4[Fucα1-2 ]GlcNAcβ1-3Galβ1-4Glc、Manα1-2Man、Manα1-3Manα1-6Man、Manα1-6Man Fucα1-2Galβ1-4Glc, Fucα1-6Glc, Xylβ1-6Glc, GlcNAcα1- 6Glc, GlcNAcβ1-4GlcNAc, GlcNAcβ1-6Glc, GlcNAcβ1-3Galβ1-4GlcNAcβ1 -3Galβ1-4Glc, GalNAcα1-6Glc, GalNAcβ1-3Gal, Neu5Acα2-3Galβ1-4Glc. Neu5Acα2-3Galβ1-4GlcNAc; α2-3Galβ1-4GlcNAcβ1-6Glc, Neu5Acα2-6Galβ1-4Glc, Neu5Acα2-6Galβ1 -3GlcNAcβ1-6Glc, Neu5Acα2-6Galβ1-4GlcNAcβ1-6Glc, GlcNS6Sα1-4Ido A2Sβ1-6Glc, Galβ1-4GlcGalβ1-4GlcNAcβ1-6Glc, Neu5Acα2-3Galβ1-4Glc Neu5Acα2-6Galβ1-4Glc, Neu5Acα2-3Galβ1-3GlcNAcβ1-6Glc, Neu5Acα2 -6Galβ1-3GlcNAcβ1-6Glc, Neu5Acα2-3Galβ1-4GlcNAcβ1-6Glc, Neu5Acα2 -6Galβ1-4GlcNAcβ1-6Glc.GlcAβ1-3GalNAc4Sβ1-6Glc 6Sβ1-6Glc, GlcA2Sβ1-3GalNAc6Sβ1-6Glc, GlcAβ1-3GalNAc4S6Sβ1-6Glc.Examples of such peptides include GlcNS6Sα1-4IdoA2Sα1-6Glc, GlcNSα1-4IdoA2Sα1-6Glc, GlcNS6Sα1-4GlcA2Sβ1-6Glc, GlcNSα1-4GlcAβ1-6Glc, αCD, βCD, and γCD.

[0033] In this specification, "Glc" stands for glucose, "Gal" stands for galactose, "Man" stands for mannose, "Fuc" stands for fucose, "Xyl" stands for xylose, "Ara" stands for arabinose, "Rha" stands for rhamnose, "NAc" stands for N-acetyl, "Ido" stands for idose, "Neu5Ac" stands for N-acetylneuraminic acid, "GlcA" stands for glucuronic acid, "IdoA" stands for iduronic acid, "CD" stands for cyclodextrin, and "S" in the chemical formula of the glycan stands for sulfonyl (sulfate group). For example, the "4S" in GlcAβ1-3GalNAc4Sβ1-6Glc stands for 4-O-sulfate.

[0034] In addition, "α" or "β" in the chemical formula of a glycan indicates that the hydroxyl group of the sugar at the reducing end is bonded to the hydroxyl group of the adjacent sugar in an α or β conformation. For example, "α1-4" indicates that the 1-hydroxyl group of the sugar at the reducing end is bonded to the 4-hydroxyl group of the adjacent sugar in an α conformation.

[0035] Preferably, the monosaccharide is selected from the group consisting of glucose, galactose, mannose, amino sugars and acidic sugars, and the polysaccharide is an oligosaccharide or polysaccharide consisting of one or more kinds selected from the group consisting of glucose, galactose, mannose, amino sugars and acidic sugars.

[0036] Monosaccharides and sugar chains may have a reducing end, a non-reducing end, or may have an alkylamine, an amino group, a carboxy group, an azide group, an alkyne group, or the like in the side chain. A "sugar chain having a reducing end" is, for example, a sugar chain in which the anomeric carbon atom is not substituted. A sugar chain having a reducing end can also be said to be a reducing sugar.

[0037] When the CLR ligand is a sugar chain containing an amino sugar or an acidic sugar, the CLR ligand portion may be bound to the linker by an amide bond via an amino group (e.g., -NH2 group) or a carboxy group (-COOH group) of the sugar chain. The above compound may contain only one type of sugar chain, or may contain a combination of two or more types of sugar chains.

[0038] The structure of the CLR ligand portion in the compound according to this embodiment is a structure in which a monosaccharide selected from the group consisting of glucose, galactose, mannose, amino sugars and acidic sugars is bound to a linker, or a structure in which an oligosaccharide or polysaccharide consisting of one or more types selected from the group consisting of glucose, galactose, mannose, amino sugars and acidic sugars is bound to a linker.

[0039] A TLR ligand is a compound or molecule that has the ability to bind to a TLR. In humans, ten types of TLRs have been identified, TLR1 to TLR10. For each TLR, there are identical or different TLR ligands.

[0040] As the TLR ligand, any ligand for any TLR can be used. As the TLR ligand, it may be a naturally occurring TLR ligand, which is a ligand derived from a microorganism, a virus, or an organism expressing a TLR, or it may be a synthetic TLR ligand. As the TLR ligand, a known ligand can be used.

[0041] Preferably, the TLR ligand portion binds to TLR4, TLR7 or TLR8 to induce TLR activity. That is, preferably, the TLR ligand portion binds to at least one selected from the group consisting of TLR4, TLR7 and TLR8. A TLR ligand having binding ability to TLR4 or TLR7 and inducing TLR activity is more preferred, and a TLR ligand having binding ability to TLR7 and inducing TLR activity by binding to TLR7 is even more preferred.

[0042] More specifically, examples of the TLR ligand include (i) monophosphoryl lipid A (formula (4)), pyrimidoindole (formula (5)), and 4-((3-nitro-5-phenoxyphenyl)amino)quinazoline-2-carboxylic acid (formula (6)), which are TLR ligands for TLR4 (hereinafter also referred to as "TLR4 ligands"); (ii) 1V209 (formula (7)), which is a TLR ligand for TLR7 (hereinafter also referred to as "TLR7 ligand"); (iii) VTX-2337 (formula (8)), which is a TLR ligand for TLR8 (hereinafter also referred to as "TLR8 ligand"); and (iv) R848 (formula (9)), which is a TLR ligand for TLR7 and TLR8. 1V209 is particularly preferred as the TLR ligand.

[0043] [ka]

[0044] Preferably, the structure of the TLR ligand portion is a structure in which 1V209 is bound to a linker. The compound according to this embodiment may contain a structure derived from one type of TLR ligand, or may contain structures derived from each of two or more types of TLR ligands. When the TLR ligand has, for example, an amino group, a hydroxy group, or a carboxy group, the TLR ligand portion may be bound to the linker via the amino group, hydroxy group, or carboxy group of the TLR ligand.

[0045] When the compound has multiple CLR ligand moieties per molecule, the linker preferably has a branched structure in which multiple CLR ligand moieties and TLR ligand moieties are each bonded to the end of the linker. For example, the structure of the compound in which the linker has a branched structure is exemplified by the following formula (1).

[0046] [ka]

[0047] In formula (1), R C has a CLR ligand moiety, and R T has a TLR ligand moiety. 1 ~p 3 are each independently an integer of 0 to 6.

[0048] The structures of the compounds according to the present embodiment are shown, for example, in the following formulas (10), (11), and (12).

[0049] [ka]

[0050] The compound according to the present embodiment can be produced by a known method. The compound may be synthesized by linking a first linker compound bound to a CLR ligand to a TLR ligand or a second linker compound bound to a TLR ligand, or may be synthesized by linking a CLR ligand to a second linker compound bound to a TLR ligand.

[0051] The first linker compound is, for example, a hydrocarbon chain or a hydrocarbon derivative chain having an amino group, a carboxy group, an azide group, or an alkyne group at one end that can be bound to a CLR ligand, and a hydrocarbon structure containing an amino group, a carboxy group, an azide group, or an alkyne group at the other end. The hydrocarbon chain and the hydrocarbon derivative chain may be a hydrocarbon chain consisting of carbon and hydrogen, or a part of the carbon or hydrogen may be substituted with another atom or a substituent. For example, the hydrocarbon derivative chain and the hydrocarbon derivative chain may have a part of the carbon-carbon bond (CC bond) that is the main chain substituted with a carbon-nitrogen bond (CN bond), a carbon-oxygen bond (CO bond), an amide bond (CO-NH bond), or the like.

[0052] The hydrocarbon structure at the other end may be a hydrocarbon structure consisting of carbon and hydrogen, or a part of the carbon or hydrogen may be replaced by other atoms or substituents. The hydrocarbon structure may be a straight chain or branched chain, may be cyclic, or may be a combination of both chain and cyclic structures.

[0053] For example, the first linker compound may have one end capable of binding to the above-mentioned CLR ligand, X 1 , the other end is Y 1 Examples of the compound include a compound having a structure represented by the following formula (13).

[0054] [ka]

[0055] In formula (13), m and n are each independently an integer of 0 to 6, 1 and Y 1 each independently has an amino group, a carboxy group, an azide group or an alkyne group at its terminal.

[0056] Preferably, the first linker compound is X 1 has an amino group, and Y 1 X has an azide group. 1When X has an amino group, the CLR ligand can be easily bound to the first linker compound via the amino group. The amino group may be an unmodified amino group, an amino group modified with an alkyl group such as a methyl group or a formyl group, or an amino group bonded to an aromatic ring. More specifically, X 1 An example of the first linker compound having an amino group at the amino acid residue includes a compound having a structure represented by the following formula (14).

[0057] [ka]

[0058] In formula (14), m and n are each independently an integer of 0 to 6; 1 each independently has an amino group, a carboxy group, an azide group or an alkyne group at its terminal.

[0059] Preferably, the CLR ligand is converted to, for example, X in formula (13) by a reductive amination reaction between the CLR ligand and the first linker compound. 1 At pH 3 to 4, which is the optimum condition for the reductive amination reaction, it is necessary that the amino group is not protonated. 1 The amino group contained in is preferably an amino group bonded to an aromatic ring in which an unshared electron pair exists on the nitrogen atom even at pH 3 to 4 due to conjugation with an aromatic ring, as shown in formula (14).

[0060] The first linker compound is, for example, Y in formula (13) and formula (14). 1 It is preferable that the first linker compound has an azide group at the end of Y. The CLR ligand and the TLR ligand can be conjugated via the azide group and the alkyne group of the second linker compound bound to the TLR ligand. In other words, 1 has an azide group, the azide group can be subjected to a click reaction with an alkyne group of the second linker compound bound to the TLR ligand. The click reaction is carried out, for example, using a copper catalyst.

[0061] The first linker compound is Y 1 The CLR ligand and the TLR ligand can be linked to each other by a click reaction via the alkyne group and an azide group of the second linker compound bound to the TLR ligand to form a complex.

[0062] The second linker compound is, for example, a hydrocarbon chain or a hydrocarbon-derived chain having an amino group, a carboxy group, an azide group, or an alkyne group at one end capable of binding to a TLR ligand, and a hydrocarbon structure containing an amino group, a carboxy group, an azide group, an alkyne group, or a group convertible to an alkyne group such as a tert-butyl group at the other end. The hydrocarbon chain and the hydrocarbon-derived chain are the same as those in the first linker compound described above.

[0063] For example, the second linker compound may have one end capable of binding to the TLR ligand as described above, which is linked to Y 2 , and the other end is X 2 Examples of the compound include a compound having a structure represented by the following formula (15).

[0064] [ka]

[0065] In formula (15), m and n are each independently an integer of 0 to 6, 2 and Y 2 Each of X independently has an amino group, a carboxy group, an azide group or an alkyne group at its terminal. 2 X has an amino group, a carboxy group, an azide group, an alkyne group or a group convertible to an alkyne group at its terminal, and may have a hydrocarbon chain or a hydrocarbon-derived chain as its main chain, which may have a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond or the like, in a linear or branched structure. 2 When the hydrocarbon chain or hydrocarbon-derived chain of X has a branched structure, the number of branches is not particularly limited. 2has a branched structure consisting of two or three chains. 2 The amino group, carboxy group, azide group or alkyne group contained in may be composed only of hydrocarbons or may be a heterocycle containing an element other than carbon.

[0066] Preferably, X 2 has a structure represented by the following formula (16) or formula (17).

[0067] [ka]

[0068] In formula (16) and formula (17), p 1 ~p 5 are each independently an integer of 0 or more and 6 or less. Z also has an amino group, a carboxy group, an azide group, an alkyne group or a group convertible to an alkyne group at its terminal, and is a hydrocarbon chain or a hydrocarbon-derived chain which may have a carbon-carbon bond, a carbon-oxygen bond or a carbon-nitrogen bond in its main chain.

[0069] X 2 When X has a branched structure, the linkers in the compound synthesized using the second linker compound have a branched structure each having a CLR ligand moiety, and the compound has multiple CLR ligand moieties per molecule. 2 The amino group, carboxy group, azide group or alkyne group contained in may be composed only of hydrocarbons or may be a heterocycle containing an element other than carbon.

[0070] Preferably, the second linker compound is X 2 has an azide group at the end of Y 2 The amino group may be an unmodified amino group, an amino group modified with an alkyl group such as a methyl group or a formyl group, or an amino group bonded to an aromatic ring. 2When the TLR ligand has an amino group, for example, a TLR ligand having a carboxy group can be easily introduced into the second linker compound via the amino group using a condensing agent or the like. An example of a TLR ligand having a carboxy group is 1V209. The condensing agent used in the condensation reaction (amidation reaction) between the TLR ligand and the second linker compound is not particularly limited, and examples thereof include HATU, COMU, PyBOP, EDC, and the like. Note that the "TLR ligand having a carboxy group" refers to a ligand that does not lose its binding affinity to TLR after binding to the second linker compound. The TLR ligand having a carboxy group may be a TLR ligand having a carboxy group at the molecular end, or may be a compound having a carboxy group introduced at the molecular end to the extent that it does not lose its binding affinity to TLR.

[0071] The second linker compound is Y 2 Y may have a carboxy group. 2 When the second linker compound has a carboxy group, a TLR ligand having, for example, an amino group can be easily introduced to the second linker compound via the carboxy group. After the TLR ligand is introduced, the carboxy group becomes a carbonyl group bound to the TLR ligand. The "TLR ligand having an amino group" is one that does not lose its binding affinity to TLR after binding to the second linker compound. The TLR ligand having an amino group may be a TLR ligand having an amino group at the molecular terminal, or may be a compound having an amino group introduced at the molecular terminal to the extent that it does not lose its binding affinity to TLR.

[0072] Preferably, X 2 has a structure represented by the following formula (18) having an alkyne group at its terminal.

[0073] [ka]

[0074] In formula (18), p 1 ~p 3 are each independently an integer of 1 or more and 6 or less.

[0075] More specifically, X 2 An example of the second linker compound having an alkyne group is a compound having a structure represented by the following formula (19).

[0076] [ka]

[0077] In formula (19), m and n each independently represent an integer of 0 to 6. 2 is Y in the above formula (15) 2 is the same as:

[0078] The second linker compound is X 2 By having an alkyne group at the end of X, the CLR ligand and the TLR ligand can be conjugated by the above-mentioned click reaction via the alkyne group and the azide group of the first linker compound bound to the CLR ligand. 2 When X has a carboxy group at its terminal, the carboxy group may be bonded to an amino group of the CLR ligand by a condensation reaction. 2 has a group convertible to an alkyne group at its terminus, the group convertible to an alkyne group may be converted to an alkyne group after the TLR ligand and the second linker compound are bonded together.

[0079] The synthetic route of the compound according to this embodiment is exemplified below. The first linker compound and the second linker compound are added to the CLR ligand and the TLR ligand as "crosslinking groups", respectively.

[0080] [ka]

[0081] In the synthetic route shown in Scheme 1, the TLR ligand moiety to which a bridging group having a carboxyl group is bound due to the introduction of a second linker compound having a terminal carboxyl group and the amino group of a sugar chain (amino sugar) serving as a CLR ligand are dehydration-condensed to link the TLR ligand moiety and the CLR ligand moiety via an acid amide bond (peptide bond). In this case, the linker is a moiety containing an acid amide bond and a bridging group.

[0082] In the synthetic route shown in Scheme 2, first, a first linker compound having an amino group at its terminal and a hydroxy group of a sugar chain (reducing sugar) serving as a CLR ligand are dehydration-condensed to add a crosslinking group to the sugar chain. Then, the TLR ligand and the sugar chain are bonded via the crosslinking group to link the TLR ligand portion and the CLR ligand portion.

[0083] In the synthetic route shown in Scheme 3, a CLR ligand moiety to which a crosslinking group having an azide group is bonded by introducing a first linker compound having an azide group at its terminal, and a TLR ligand moiety to which an alkyne group is bonded by introducing a second linker compound having an alkyne group at its terminal are subjected to a click reaction, thereby linking the TLR ligand moiety and the CLR ligand moiety.

[0084] The above-mentioned compound may be contained in the immune enhancing agent according to the present embodiment in the form of a salt. The salt of the compound is not particularly limited as long as it exhibits immune enhancing activity and is a pharmacologically acceptable salt. The salt is not particularly limited, and examples thereof include alkali metal salts such as lithium salt, sodium salt, and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, oxalate, and phosphate; and acetate, propionate, hexanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, fumarate, tartrate, citrate, benzoate, o-(4-hydroxybenzoyl)benzoate, cinnamate, mandelate, methanesulfonate, ethoxylate, ethyl acetate, ethyl methacrylate, ethyl acrylate, ethyl meth ... Examples of organic acid salts include benzenesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, 2-naphthalenesulfonate, p-toluenesulfonate, camphorsulfonate, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylate, glucoheptanoate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, and muconate.

[0085] The immune enhancing agent according to the present embodiment is produced by a known method and contains 0.000001 to 99.9 mass%, 0.00001 to 99.8 mass%, 0.0001 to 99.7 mass%, 0.001 to 99.6 mass%, 0.01 to 99.5 mass%, 0.1 to 99 mass%, 0.5 to 60 mass%, 1 to 50 mass%, or 1 to 20 mass% of the above compound as an active ingredient. The immune enhancing agent may be a solid preparation or a liquid preparation.

[0086] In addition to the above-mentioned compound, the immune enhancing agent may contain any pharmacologically acceptable component. The optional components include, for example, a carrier, an excipient, a lubricant, a binder, a disintegrant, a solvent, a solubilizing agent, a suspending agent, an isotonic agent, a buffer, a soothing agent, etc. Furthermore, additives such as a preservative, an antioxidant, a coloring agent, and a sweetener may be added to the immune enhancing agent as necessary.

[0087] The dosage of the immune enhancing agent according to the present embodiment is appropriately determined depending on the sex, age, weight, symptoms, antigen, etc. of the subject to be administered. The immune enhancing agent is administered so that the compound is in an effective amount. An effective amount is the amount of the compound required to obtain the desired result, that is, the amount required to bring about immune enhancing activity.

[0088] The dosage of the immune enhancer is, for example, 0.01 mg / kg to 1000 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, more preferably 0.2 mg / kg to 20 mg / kg, and can be administered once a day or in divided doses of more than one. When the immune enhancer is administered in divided doses, the immune enhancer is administered 1 to 4 times a day. The immune enhancer may also be administered at various administration frequencies, such as daily, every other day, once a week, every other week, or once a month. If necessary, an amount outside the above range can also be used.

[0089] The administration route of the immune enhancer is not particularly limited. The immune enhancer may be administered, for example, parenterally or orally. In the case of parenteral administration, it may be intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, transmucosal administration, pulmonary administration, enteral administration, etc. The immune enhancer may be administered via infusion.

[0090] The immune enhancer can be in any form of formulation. In the case of oral administration, the immune enhancer may be in the form of tablets such as sugar-coated tablets, buccal tablets, coated tablets, chewable tablets, etc.; liquids such as troches, pills, powders, capsules including soft capsules, granules, suspensions, emulsions, syrups including dry syrups, and elixirs. In the case of parenteral administration, the immune enhancer may be in the form of injections, inhalants, transdermal tapes, aerosols, suppositories, etc.

[0091] The immune enhancing agent is administered to any subject. For example, the subjects to which the immune enhancing agent is administered are humans and non-human animals, preferably mammals. The subjects include, for example, primates such as humans and chimpanzees, experimental animals such as rats, mice, and rabbits, livestock animals such as pigs, cows, horses, sheep, and goats, and pet animals such as dogs and cats.

[0092] The immune enhancing agent according to the present embodiment has high translocation ability to immune cells and can activate immune cells more effectively. Since the immune enhancing agent has a CLR ligand portion and a TLR ligand portion, it has an effect of enhancing natural immune responses, as shown in the following examples.

[0093] Furthermore, as shown in the following Test Example 3, the immune enhancing agent according to the present embodiment has a protective effect against viral infections, and therefore can also be used as an antiviral drug.

[0094] In addition, since the compound may be metabolized in vivo, the CLR ligand moiety and the TLR ligand moiety may dissociate and bind to the CLR and the TLR, respectively, thereby inducing or enhancing an immune response. Furthermore, after the compound is transported to an immune cell, the CLR ligand moiety and the TLR ligand moiety may dissociate.

[0095] Furthermore, as shown in the following Test Examples 2 to 7, the immune enhancer according to the present embodiment is also useful as a mucosal immune enhancer administered via the mucosa. The site of administration of the mucosal immune enhancer is not particularly limited as long as it is the mucosa. Preferably, the mucosal immune enhancer is administered from the nasal mucosa by a known method. The mucosal immune enhancer can efficiently transport antigens to immune cells present under the mucosal tissue and activate the immune cells. Since the mucosal immune enhancer contains a compound having a CLR ligand portion and a TLR ligand portion, it can more effectively induce natural immune responses mediated by CLR and TLR.

[0096] The immune enhancing agent according to the present embodiment may be a vaccine adjuvant. The vaccine adjuvant may contain a solvent in addition to the above-mentioned compound. Examples of the solvent include water, physiological saline, and glycerol. The solvent can be appropriately selected in consideration of the solubility of the above-mentioned compound, the reaction rate, and the like.

[0097] The content of the solvent in the vaccine adjuvant is not particularly limited, but for example, 1 to 500 parts by mass or 20 to 200 parts by mass of the solvent per part by mass of the compound. The vaccine adjuvant may contain other components other than the above-mentioned compound and solvent as necessary. The other components are not particularly limited as long as they do not interfere with the induction of an immune response by the vaccine adjuvant, but examples thereof include a buffer, a surfactant, a desiccant, a preservative, and a mucoadhesive.

[0098] The vaccine adjuvant according to the present embodiment is administered to a subject in combination with an antigen. The antigen may be a bacterial antigen, a virus antigen, a protein such as a toxoid, a glycoprotein, an allergen, a carbohydrate, a nucleic acid, or the like, which is purified for vaccines or is commercially available. The antigen may be, for example, an antigen for a vaccine against chickenpox virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, coronavirus, West Nile virus, hantavirus, dengue virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, HIV virus, hepatitis C virus, swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, bovine viral diarrhea virus, bovine infectious lymphoma virus, Bordetella pertussis, Neisseria meningitidis, Haemophilus influenzae type b, Pneumonia bacillus, Vibrio cholerae, malaria pathogens, or the like. The antigen may preferably be HA, which is a spike protein of influenza virus, or the like, capsular polysaccharides of pneumococcus and influenza virus, or the like. The antigen may be an inactivated or attenuated antigen. The antigen may be one type or multiple types.

[0099] The vaccine adjuvant according to the present embodiment is also useful as a vaccine for cancer immunotherapy. When used as a vaccine adjuvant in a vaccine for cancer immunotherapy, the antigen is, for example, a gene or protein (cancer antigen) that is specifically expressed in cancer cells or overexpressed compared to normal cells. Examples of cancer antigens include MAGEA1-4, NY-ESO-1, PRAME, SSX2, CT83, CD19, GP100, MART1, PSA, PSMA, tyrosinase, WT1, HER2, MUC1, CEA, survivin, cyclin B1, EGFR, mesothelin, telomerase, MUC1T, LSP1, BCR-ABL1, HRAS, KRAS, and the like. The vaccine for cancer immunotherapy may be a peptide vaccine, a gene vaccine, or a dendritic cell (DC) vaccine.

[0100] The term "combination" includes not only administering a vaccine composition containing an immunopotentiator, which is a vaccine adjuvant, and an antigen to a subject, but also administering the immunopotentiator and the antigen separately and simultaneously, or administering them separately and consecutively. The route of administration of the antigen may be the same as the route of administration of the immunopotentiator, or may be a different route.

[0101] The site of administration of the vaccine adjuvant is not particularly limited. Preferably, the vaccine adjuvant is administered by a known method. The dose of the vaccine adjuvant is appropriately determined depending on the sex, age, weight, symptoms, type of antigen, etc. of the subject. The vaccine adjuvant is administered so that the compound is an effective amount. The effective amount is the amount of the compound necessary to obtain the desired result, and in particular, the amount capable of inducing an immune response.

[0102] The mucosal immune enhancer according to the present embodiment is also useful as an adjuvant for mucosal vaccines.

[0103] In another embodiment, a vaccine composition is provided. The vaccine composition comprises an immunopotentiator that is an adjuvant for a vaccine according to the above embodiment, and an antigen. In another embodiment, a mucosal vaccine composition is provided. The mucosal vaccine composition comprises an immunopotentiator that is an adjuvant for a mucosal vaccine according to the above embodiment, and an antigen. Preferably, the mucosal vaccine composition is for administration via the nasal mucosa.

[0104] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0105] [Production Example 1: Synthesis of a single-chain linker component having 1V209] As shown below, the 1V209 single-chain derivative (4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)-N-(prop-2-yn-1-yl)benzamide, formula (20)) was synthesized in 62% yield by condensation reaction of 1V209 with propargylamine (second linker compound).

[0106] [ka]

[0107] In detail, 1V209 (104 mg, 288 μmol) and HATU (190 mg, 500 μmol) were dissolved in DMF (5 mL) under an argon atmosphere, propargylamine (47.6 mg, 864 μmol) was added, and the mixture was stirred at 0 °C for 24 hours. Chloroform and distilled water were added to the reaction solution, and the mixture was extracted with chloroform. The organic layer was washed once with distilled water and once with saturated saline, and dried over anhydrous magnesium sulfate. The desiccant was removed by filtration, and the mixture was concentrated under reduced pressure using an evaporator. The residue was purified by medium pressure silica gel chromatography (chloroform:methanol = 20:1) to obtain 1V209 single-chain derivative (formula (20)) as a white solid (71.0 mg, 62%). 1V209 was synthesized according to the synthetic route described in the literature (Michael Chan, et al., Synthesis and Characterization of PEGylated Toll Like Receptor 7 Ligands, Bioconjugate Chem., 22 (2011) 445-454).

[0108] 1 H-NMR(400MHz,DMSO(d6))δ10.03(1H,s),8.90(1H,dd,J=5.5,5.5Hz),7.81(2H,d,J=8.2Hz),7.37(2H,d,J=8.2Hz),6.51(2H,s),4.9 2(2H,s),4.26(2H,dd,J=5.5,4.1Hz),4.05(2H,dd,J=5.5,2.7Hz),3.58(2H,t,J=4.6Hz),3.12(1H,dd,J=2.5Hz),2.52-2.50(1H,m). 13 C-NMR(100MHz,DMSO(d6))δ165.6,159.8,152.2,149.1,147.7,140.4,132.9 ,128.6,127.5,127.3,120.4,98.3,81.3,72.8,70.2,65.3,58.1,42.1,28.4. ESI-TOF / MS(positive mode);Found:m / z 397.1624[(M+H) + ],Calcd.For C 19 H 20N6O4:397.1619.

[0109] [Production Example 2: Synthesis of a three-branched linker component having 1V209] As shown below, the 1V209 tribranched derivative (4-(2-(4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)benzamido)acetamido)-4-(3-oxo-3-(prop-2-yn-1-ylamino)propyl)-N1,N7-di(prop-2-yn-1-yl)heptanediamide, formula (21)) was synthesized in 58% yield. For the 1V209 tribranched derivative, the benzyloxycarbonyl (Z) compound (a) synthesized by the synthetic route described in the literature (Yasuo Suda, et al., Immobilization and Clustering of Structurally Defined Oligosaccharides for Sugar Chips: An Improved Method for Surface Plasmon Resonance Analysis of Protein-Carbohydrate Interactions, Bioconjugate Chem., 17 (2006) 1125-1135) was converted to the amine compound (b) as the second linker compound by catalytic reduction, and then condensed with 1V209 using HATU (c). Next, the tert-butyl group was removed by treatment with TFA, and the condensation reaction was carried out using HATU and propargylamine.

[0110] [ka]

[0111] In detail, 1V209 (71.8 mg, 200 μmol) and HATU (116 mg, 300 μmol) were dissolved in DMF (5 mL) under an argon atmosphere, the amine was added, and the mixture was stirred for 12 hours under ice cooling. Chloroform and distilled water were added to the reaction solution, and the mixture was extracted into a chloroform layer. The organic layer was washed once with distilled water and once with saturated saline, and dried over anhydrous magnesium sulfate. The desiccant was removed by filtration, and the mixture was concentrated under reduced pressure using an evaporator. The residue was purified by medium pressure silica gel chromatography (chloroform:methanol = 20:1) to obtain a yellow solid (119 mg, 75%).

[0112] 1 H-NMR (600MHz, CDCI3) δ7.83(2H,d),7.50(2H,d),5.04(2H,s),4.42(2H,t),3.72(2H,t),3.40(3H,s),2.22(6H,t),1.96(6H,t),1.41(27H,s). 13 C-NMR (100MHz, CDCI3) δ172.7,172.6,170.1,167.9,160.5,153.9,149.4,147.9,140.4,132. 6,128.6,127.9,99.4,80.8,80.6,70.7,66.2,59.0,58.2,39.8,30.0,29.8,29.6,28.1,28.0. ESI-TOF / MS(positive mode);Found:m / z 814.4345[(M+H) + ],Calcd.for C 40 H 60 N7O 11 :814.4345.

[0113] The above yellow solid (115 mg, 141 μmol) was dissolved in a solution of TFA:CH2CI2:H2O=1:1:0.1 and stirred for 24 hours. After that, the mixture was concentrated under reduced pressure to quantitatively obtain a white solid (115 mg). The obtained white solid (47.1 mg, 73.0 μmol) and HATU (90.1 mg, 237 μmol) were dissolved in DMF (3 ml), propargylamine (27.0 mg, 490 μmol) was added, and the mixture was stirred under ice cooling for 24 hours. Chloroform and distilled water were added to the reaction solution, and the mixture was extracted into a chloroform layer. The organic layer was washed once with distilled water, once with a saturated sodium bicarbonate solution, and once with saturated saline, and then dried over anhydrous magnesium sulfate. The drying agent was removed by filtration, and the mixture was concentrated under reduced pressure using an evaporator. The residue was purified by medium pressure silica gel chromatography (chloroform:methanol=20:1) to obtain the 1V209 three-branched derivative (formula (21)) as a yellow solid (21.4 mg, 58%).

[0114] 1 H-NMR(400MHz,DMSO(d6))δ8.31(3H,dd,J=5.5,2.7Hz),7.85(2H,d,J=8.2Hz),7.36(3H,d,J=8.2Hz),6.80(2H,s),4.91(2H,s),4.25( 2H,t,J=5.0Hz),3.82(6H,dd,J=5.5,2.3Hz),3.58(2H,dd,J=6.0,3.0Hz),3.08(3H,t,J=2.5Hz),2.06-2.03(5H,m),1.82-1.79(6H,m). 13 C-NMR(100MHz,DMSO(d6))δ171.9,168.1,166.3,159.9,152.1,149.0,147.9,140.4,133.1,12 7.6,127.2,98.5,81.3,79.4,79.6,77.6,72.9,70.2,65.3,58.1,56.9,30.0,29.3,28.3,27.9. ESI-TOF / MS(positive mode);Found:m / z 779.3236[(M+Na) + ],Calcd.for C 37 H 44 N 10 O8Na:779.3236.

[0115] [Production Example 3: Synthesis of azidoamine] As shown below, azidoamine (N-(3-aminophenyl)-2-azidoacetamide, formula (22)) as the first linker compound was synthesized in 60% yield by the condensation reaction of m-phenylenediamine with monochloroacetic acid and the substitution reaction of the chloro group with azide ion.

[0116] [ka]

[0117] In detail, m-phenylenediamine (211 mg, 2.24 mmol) and monochloroacetic acid (240 mg, 2.22 mmol) were reacted with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (427 mg, 2.42 mmol) as a condensation agent in N,N-dimethylformamide (DMF, 10 mL) under ice cooling for 4 hours. Sodium azide (281 mg, 4.31 mmol) was then added and the mixture was stirred at 60°C for 24 hours. Distilled water was added to stop the reaction, and the mixture was washed once with distilled water and once with saturated saline, and dried over anhydrous magnesium sulfate. The drying agent was removed by filtration, and the mixture was concentrated using an evaporator. The residue was dissolved in a chloroform-methanol solution and adsorbed onto a small amount of silica gel. Purification by medium pressure silica gel chromatography (toluene:ethyl acetate=3:2) gave the azidoamine (formula (22)) as a yellow solid (260 mg, 60%).

[0118] 1 H-NMR(400MHz,CD3OD)δ6.92(2H,dd,J=10.7,4.9Hz),6.70(1H,d,J=7.3Hz),6.38(1H,d,J=7.3Hz),3.84(2H,s). 13 C-NMR(400MHz,CD3OD)δ168.2,149.4,139.8,130.4,112.9,111.1,108.4,53.2. ESI-TOF / MS(positive mode);Found:m / z 214.0643[(M+Na) + ],Calcd.for C8H9N5ONa:214.0699.

[0119] [Production Example 4: Synthesis of sugar chains having azide groups] As shown below, αGlc4-N3 (2-azido-N-({[(α-D-glucopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)]amino}phenyl)acetamide, formula (23)) was prepared in 83% yield by reductive amination of azidoamine (formula (22)) with glycan components. In the synthesis of αGlc4-N3, maltose and azidoamine were mixed, and then reductive amination was carried out using sodium cyanoborohydride.

[0120] [ka]

[0121] In detail, maltose (103 mg, 284 μmol) was dissolved in distilled water (4 mL), acetic acid (0.8 mL) was added to adjust the pH to 4-5, and azidoamine (16) (55.6 mg, 290 μmol) dissolved in methanol (4 mL) was added and stirred at 50 °C for 6 h. Sodium cyanoborohydride (18.2 mg, 2.90 mmol) was then added and stirred for 24 h. The solution was concentrated under reduced pressure to recover the residue, which was purified by medium-pressure ODS column chromatography (distilled water:methanol = 5:5) to give αGlc4-N3 as a white solid (118 mg, 83%).

[0122] 1H-NMR(600MHz,D2O)δ7.18(1H,dd,J=8.4,8.4Hz),6.86(1H,s),6.74(1H,d,J=8.4Hz),6.61(1H,d,J=8.4Hz), 5.03(1H,d,J=4.8Hz),4.06(2H,s),3.93-3.91(1H,m),3.88(1H,dt,J=7.5,4.0Hz),3.84-3.80(3H,m),3.77(1 H,dd,J=12.2,2.0Hz),3.71(1H,dd,J=12.2,4.0Hz),3.67-3.66(2H,m),3.56(1H,dd,J=12.2,7.5Hz),3.49(1 H,dd,J=9.9,4.0Hz),3.37(1H,dd,J=4.0,9.5Hz),3.27(1H,dd,J=13.6,4.8Hz),3.17(1H,dd,J=13.6,8.2Hz). 13 C-NMR(150MHz,D2O)δ169.6,149.2,137.9,130.7,112.6,112.4,108.0,101.1,82.6,7 3.5,73.1,73.0,72.2,71.9,69.9,69.5,62.7,60.9,52.8,47.1.ESI-TOF / MS(positive mode);Found:m / z 540.1907[(M+Na) + ],Calcd.for C 20 H 31 N5O 11 Na:540.1912.

[0123] The following azide-containing oligosaccharides, which have different glycan components from αGlc4-N3, were synthesized by the same procedure as for αGlc4-N3, except for the different glycan components. βGlc6-N3(2-azido-N-({[(β-D-glucopyranosyl)-(1→6)-(1-deoxy-D-glucitol-1-yl)]amino}phenyl)acetamide, yield 84%) 1H-NMR(400MHz,D2O-DMSO(d6))δ7.07(1H,t,J=7.8Hz),6.78(1H,dd,J=1.8,2.3Hz),6.67( 1H,dd,J=2.3,8.4Hz), 6.48(1H,dd,J=1.8,8.2Hz),4.23(1H,d,J=7.8Hz),3.95(2H,s),3 2(1H,dd,J=2.7,11.4Hz),3.83-3.56(6H,m),3.51(1H,dd,J=4.6,11.9Hz),3.29(1H,dd,J= 8.7,9.1Hz), 3.24-3.14(3H,m), 3.12(1H,dd,J=8.7,9.1Hz),2.97(1H,dd,J=8.2,13.7Hz). 13 C-NMR(100MHz,D2O)δ170.0,150.3,139.1,131.6,112.8,112.6,109.0. 104.4,77.3,77.1,74.7,72.9,72.6,72.0,71.3,71.0,62.1,53.6,47.5 ESI-TOF / MS(positive mode);Found:m / z 540.1907[(M+Na) + ],Calcd 20 H 31 N5O 11 In:540.1912.

[0124] βGlc4-N3(2-azido-N-({[(β-D-glucopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)]amino}phenyl)acetamide、Concentration: 88%) 11H-NMR (600 MHz, D2O-DMSO(d6)) δ 7.06 (1H, ddd, J = 2.0, 7.5, 8.2 Hz), 6.78 (1H, d, J = 2.0 Hz), 6.65 (1H, dd, J = 2.0, 7.5 Hz), 6.50 (1H, dd, J = 2.0, 8.2 Hz), 4.35 (1H, d, J = 8.2 Hz), 3.95 (2H, s), 3.92 - 3.87 (1H, m), 3.76 - 3.70 (2H, m), 3.68 - 3.63 (2H, m), 3.57 - 3.51 (2H, m), 3.47 (1H, dd, J = 4.1, 12.2 Hz), 3.27 (1H, ddd, J = 1.4, 9.5, 10.2 Hz), 3.24 - 3.18 (2H, m), 3.17 - 3.08 (2H, m), 2.95 (1H, dd, J = 8.2, 12.2 Hz). 13 13C-NMR (150 MHz, D2O-DMSO(d6)) δ 169.3, 150.0, 138.1, 131.0, 112.3, 107.7, 109.0, 103.2, 80.0, 76.3, 76.1, 74.90, 71.5, 71.2, 71.0, 70.1, 62.6, 60.9, 52.8, 46.9. ESI-TOF / MS (positive mode); Found: m / z 540.1902 [(M+Na) + , Calcd. for C 20 H 31 N5O 11 Na: 540.1912

[0125] βGal4-N3 (2-azido-N-({[(β-D-galactopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)]amino}phenyl)acetamide, Yield: 91%) 1H-NMR(600MHz, D2O-DMSO(d6)) δ 7.28(1H, t, J = 8.4Hz), 6.96(1H, s), 6.86(1H, d, J = 8.4Hz), 6.72(1H, d, J = 8.4Hz), 4.51(1H, d, J = 7.5Hz), 4.14(2H, s), 4.12(1H, t, J = 3.9Hz), 3.97 - 3.95(1H, m), 3.93(1H, dd, J = 10.2, 3.4Hz), 3.90 - 3.87(2H, m), 3.76(1H, dd, J = 11.9, 6.4Hz), 3.67(1H, dd, J = 6.5, 6.1Hz), 3.64(1H, dd, J = 3.4, 3.2Hz), 3.62(1H, d, J = 6.1Hz), 3.58(1H, dd, J = 9.5, 7.0Hz), 3.40(1H, dd, J = 12.9, 4.7Hz), 3.16(1H, dd, J = 12.9, 8.2Hz). 13 C-NMR(150MHz, D2O-DMSO(d6)) δ 169.5, 149.5, 137.9, 130.6, 112.6, 112.5, 108.1, 103.7, 80.2, 75.6, 73.1, 71.7, 71.6, 71.1, 70.6, 69.1, 62.6, 61.3, 52.8, 47.0. ESI-TOF / MS; Found: m / z 540.1912[(M + Na) + , Calcd. For C 20 H 31 N5O 11 Na: 540.1912.

[0126] αGal6-N3(2-azido-N-({[(α-D-galactopyranosyl)-(1→6)-(1-deoxy-D-glucitol-1-yl)]amino}phenyl)acetamide, Yield: 54%) ESI-TOF / MS; Found: m / z 540.1902[(M + Na) + , Calcd. For C 20 H 31 N5O 11 : 540.1912.

[0127] αFuc2-βGal4-N3(2-azido-N-({[(α-D-fucosyl)-(1→2)-(β-D-galactopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)]amino}phenyl)acetamide、Concentration: 85%); 1 H-NMR(400MHz,D2O-DMSO(d6))δ7.11(1H,dd,J=7.9,8.2Hz),6.79(1H,dd,J=1.8,2.3Hz),6.70(1H,dd,J=1.8,2.3Hz), J=1.8,7.9Hz), 6.56(1H,dd,J=2.3,8.2Hz),5.16(1H,d,J=3.2Hz),4.42(1H,d,J=8.2Hz),4.09(1H,dd, J=5.9,12.8Hz),4.00(2H,s),3.84(1H,ddd,J=4.9,4.8,8.8Hz),3.99-3.93(1H,m),3.79-3.60(10H,m) ,3.57-3.46(5H,m),3.25(1H,dd,J=3.7,12.8Hz),2.98(1H,dd,J=8.2,13.6Hz),1.04(3H,d,J=6.4Hz). ESI-TOF / MS(positive mode);Found:m / z 664.2672[(M+Na) + ],Calcd 20 H 31 N5O 11 At:664.2678.

[0128] αXyl6-N3(2-azido-N-({[(α-D-xylopyranosyl)-(1→6)-(1-deoxy-D-glucitol-1-yl)]amino}phenyl)acetamide、Concentration: 74%) 11H-NMR (400 MHz, D2O-DMSO(d6)) δ 7.11 (1H, dd, J = 8.2, 8.2 Hz), 6.79 (1H, dd, J = 1.4, 2.0 Hz), 6.69 (1H, dd, J = 1.4, 8.2 Hz), 6.54 (1H, dd, J = 2.0, 8.2 Hz), 4.71 (1H, d, J = 3.4 Hz), 4.00 (2H, s), 3.84 (1H, ddd, J = 4.9, 4.8, 8.8 Hz), 3.77 (1H, ddd, J = 2.7, 4.8, 7.5 Hz), 3.74 - 3.69 (2H, m), 3.63 (1H, dd, J = 2.7, 8.2 Hz), 3.55 - 3.48 (2H, m), 3.48 - 3.39 (3H, m), 3.38 (1H, dd, J = 4.1, 9.5 Hz), 3.24 (1H, dd, J = 4.8, 13.6 Hz), 3.02 (1H, dd, J = 7.5, 13.6 Hz). 13 13C-NMR (100 MHz, D2O-DMSO(d6)) δ 170.0, 150.3, 139.1, 131.6, 112.8, 112.6, 109.0, 104.4, 77.3, 77.1, 74.7, 72.9, 72.6, 72.0, 71.3, 71.0, 62.1, 53.6, 47.5 ESI-TOF / MS (positive mode); Found: m / z 510.1807 [(M+Na) + , Calcd. for C 19 H 29 N5O 10 Na: 510.1807

[0129] α-Ara5-N3 (2-azido-N-({[(α-D-arabinopyranosyl)-(1→5)-(1-deoxy-D-arabinitol-1-yl)]amino}phenyl)acetamide, Yield: 65%) 1H-NMR(600MHz, D2O-DMSO(d6)) δ 7.10(1H, dd, J = 8.2Hz), 6.80(1H, dd, J = 1.3, 2.0Hz), 6.69(1H, dd, J = 1.3, 8.2Hz), 6.51(1H, dd, J = 2.0, 8.2Hz), 4.88(1H, brs), 4.00(2H, s), 3.96 - 3.87(3H, m), 3.78(1H, dd, J = 2.7, 6.1Hz), 3.74 - 3.70(2H, m), 3.65(1H, dd, J = 3.4, 12.9Hz), 3.60(1H, dd, J = 4.8, 13.6Hz), 3.54(1H, dd, J = 4.8, 12.9Hz), 3.47(1H, dd, J = 1.3, 7.5Hz), 3.18(1H, dd, J = 4.8, 13.6Hz), 3.10(1H, dd, J = 8.2, 13.6Hz). 13C-NMR(150MHz, D2O-DMSO(d6)) δ 169.7, 150.4, 138.8, 131.6, 113.8, 112.8, 108.9, 102.1, 85.3, 82.4, 78.0, 72.4, 70.8, 70.4, 69.1, 62.7, 56.5, 53.6. ESI-TOF / MS(positive mode); Found: m / z 480.1701[(M + Na) + , Calcd. for C 18 H 27 N5O9Na: 480.1701

[0130] α-Man6-N3(2-azido-N-(α-D-mannopyranosyl)-(1→6)-(1-deoxy-D-glucitol-1-yl)amino(phenyl)acetamide, Yield: 84%) 1H-NMR(600MHz,D2O)δ7.09(1H,t,J=8.4Hz),6.77(1H,s),6.67(1H,d,J=7.5Hz),6.53(1H,d,J= 8.2Hz), 3.97(2H,s),3.83(1H,ddd,J=7.8,7.8,4.0Hz),3.80(1H,dd,J=4.0,1.4Hz),3.75(2H,t t,J=8.2,2.6Hz),3.71(1H,s),3.71-3.67(3H,m),3.66(1H,d,J=2.6Hz),3.61-3.58(2H,m),3 54-3.52(1H,m), 3.50-3.47(2H,m), 3.20(1H,dd,J=13.6,4.1Hz),3.00(1H,dd,J=13.6,7.8Hz). 13 C-NMR(150MHz,D2O)δ169.6,149.2,137.9,130.7,112.6,112.4,108.0,101 .1,82.6,73.4,73.0,73.0,72.2,71.9,69.9,69.5,62.7,60.9,52.8,47.1. ESI-TOF / MS(positive mode);Found:m / z 540.1907[(M+Na) + ],Calcd. for C 20 H 31 N5O 11 In:540.1912.

[0131] (βGlcNAc4)4-N3(2-azido-N-[(2-acetamido-1-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-1-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetoamido-1-deoxy- β-D-glucopyranosyl)-(1→4)-(2-acetamido-1-deoxy-β-D-glucopyranosyl)-(1→4)-(2-acetamido-1,2-deoxy-D-glucitol-1-yl)amino(phenyl)]acetamide、Concentration:58%); 1H-NMR (600 MHz, CD3OD) δ 7.19 (1H, dd, J = 8.4, 8.4 Hz), 6.93 (1H, s), 6.80 (1H, d, J = 8.4 Hz), 6.61 (1H, d, J = 8.4 Hz), 4.57 - 4.51 (4H, m), 4.31 (1H, dd, J = 12.2, 6.7 Hz), 4.11 (3H, d, J = 7.8 Hz), 3.90 (2H, d, J = 12.2 Hz), 3.83 (2H, d, J = 10.1 Hz), 3.76 - 3.74 (4H, m), 3.70 - 3.55 (13H, m), 3.53 - 3.51 (6H, m), 3.41 (1H, t, J = 9.2 Hz), 3.36 - 3.33 (1H, m), 3.22 (1H, dd, J = 14.3, 7.5 Hz), 2.04 (3H, s), 2.04 (3H, s), 2.04 (3H, s), 2.01 (3H, s), 1.97 (3H, s). 13 C-NMR (150 MHz, CD3OD) δ 174.6, 169.0, 130.8, 115.2, 111.9, 111.5, 107.0, 104.3, 104.0, 86.2, 86.2, 80.9, 77.5, 77.1, 77.0, 75.8, 74.9, 74.6, 74.5, 74.5, 72.0, 71.9, 71.8, 71.0, 69.9, 64.1, 62.1, 62.0, 53.2, 49.6, 49.4, 49.3, 49.0, 49.0, 48.9, 48.7, 48.6, 46.8. ESI-TOF / MS (positive mode); Found: m / z 1231.4810 [(M + Na) + , Calcd. for C 48 H 76 N 10 O 26 Na: 1231.4824.

[0132] (βGlc3)4-N3(2-azido-N-[(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(1-deoxy-D-glucitol-1-yl)amino(phenyl)]acetamide, Yield: 52%) 1 H-NMR(600MHz,CD3OD)δ7.16(1H,dd,J=8.4,8.4Hz),6.94(1H,s),6.84(1H,d,J=8.4Hz),6.59(1H,d,J=8.4Hz),4.81-4.78(5H,m),4.74-4.69(3H,m),4.25-4.21(1H,m),4.07(2H,s),4.04(1H,d,J=6.6Hz),4.00-3.94(5H,m),3.83(1H,dd,J=11.6,2.7Hz),3.71-3.61(10H,m),3.56(1H,t,J=8.5Hz),3.51(2H,m),3.47-3.39(10H,m),3.43-3.38(2H,m),3.16(1H,dd,J=13.6,8.2Hz). 13 C-NMR(600 MHz, CD3OD)δ174.6,169.0,130.8,115.2,111.9,111.5,107.0,104.3,104.0,86.2,86.2,80.9,77.5,77.1,77.0,75.8,74.9,74.6,74.5,74.4,72.0,71.9,71.8,71.0,69.9,64.1,62.1,62.0,53.2,49.6,49.4,49.3,49.1,49.0,48.9,48.7,48.6,46.8. ESI-TOF / MS(positive mode);Found:m / z 1026.3483[(M+Na) + ],Calcd.for C 38 H 61 N5O 26 Na:1026.3497.

[0133] (αGlc6) n -N3(2-azido-N-(dextran)amino(phenyl)]acetamide、MW17,000) 1 H-NMR(400MHz,D2O)δ7.24(1H,dd,J=7.8,8.2Hz),6.89(1H,brs),6.85(1H,d,J=8.2Hz),6.69(1H,d,J=7.8Hz),5.04-4.90(~75H,m),4.10-3.36(~450H,m).

[0134] (αGlc6) n -N3(2-azido-N-(dextran)amino(phenyl)]acetamide, MW40,000) 1 H-NMR(400MHz,D2O)δ7.15-7.05(1H,br),6.82-6.67(2H,m),6.59-6.52(1H,m),4.92-4.74(~150H,m),3.97-3.22(~900H,m).

[0135] [Production Example 5: Synthesis of glycan-TLR ligand conjugate by click reaction] The glycan-TLR ligand conjugate αGlc4-1V209 (4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)-N-(α-D-glucopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)amino(phenyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide, formula (10), was synthesized as shown below. In the synthesis of αGlc4-1V209, αGlc4-N3 (formula (23)) and the 1V209 single-chain derivative (formula (20)) were mixed, and the click reaction was carried out in the presence of copper sulfate and sodium ascorbate to prepare αGlc4-1V209 (formula (10)) in 49% yield.

[0136] [ka]

[0137] Specifically, αGlc4-N3 (15.2 mg, 38.3 μmol) was dissolved in DMF (1 mL), copper sulfate pentahydrate (4.78 mg, 19.2 μmol) and sodium ascorbate were added, and the 1V209 single-chain derivative (αGlc4-1V209, 14.2 mg, 27.4 μmol) dissolved in distilled water (1 mL) was added and stirred at 50°C for 24 h. The residue was concentrated using an evaporator and purified by medium-pressure ODS column chromatography (distilled water:methanol = 5:5) to give αGlc4-1V209 as a white solid (12.2 mg, 49%).

[0138] 1 H-NMR(600MHz,D2O-DMSO(d6))δ7.97(1H,s),7.83(2H,d,J=8.2Hz),7.37(2H,d,J=8.2Hz),7.00(1H,dd,J=8.2Hz),6.86(1H,s),6.74 (1H,d,J=8.2Hz),6.35(1H,d,J=8.2Hz),5.24(2H,s),4.93(2H,s),4.85(1H,d,J=4.1Hz),4.53(2H,s),4.25(2H,dd,J=6.1,4.7Hz),3. 73(3H,dd,J=12.2,6.5Hz),3.62(3H,d,J=9.5Hz),3.57(2H,t,J=4.4Hz),3.51(1H,dd,J=12.2,3.4Hz),3.47(1H,dd,J=11.9,5.8Hz), 3.41(2H,dt,J=11.9,9.5Hz),3.28(1H,dd,J=9.5,4.1Hz),3.25(3H,s),3.13-3.11(2H,m),3.01(1H,dd,J=12.6,6.5Hz),1.97(2H,s). 13C-NMR(150MHz,D2O-DMSO(d6))δ170.5,166.4,164.1,160.2,152.6,149. 7,149.4,148.0,145.2,140.7,139.2,133.5,132.0,129.6,127.9,127.6 ,124.9,108.5,107.2,103.1,101.4,101.3,98.4,84.4,73.5,73.4,72.3 ,72.2,71.6,70.5,70.0,68.9,65.7,62.5,60.8,58.4,52.4,46.6,42.4. ESI-TOF / MS(positive mode);Found:m / z 936.3436[(M+Na) + ],Calcd.for C 39 H 51 N 11 O 15 Na:936.3458.

[0139] The following glycan-TLR ligand conjugates, which have different glycan components from αGlc4-1V209, were synthesized using the same procedures as for αGlc4-1V209, except for the glycan.

[0140] βGal4-1V209(4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)-N-(β-D-galactopyrano syl)-(1→4)-(1-deoxy-D-glucitol-1-yl)amino(phenyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide, yield: 87%) 1H-NMR(600MHz, D2O (1 drop) - DMSO(d6) (1:40)) δ 7.93(1H, s), 7.79(2H, d, J = 8.4Hz), 7.33(2H, d, J = 8.4Hz), 6.96(1H, dd, J = 8.4Hz), 6.81(1H, s), 6.72(1H, d, J = 8.2Hz), 6.33(1H, d, J = 8.2Hz), 5.21(2H, s), 4.89(2H, s), 4.49(2H, s), 4.26(1H, d, J = 7.5Hz), 4.22(2H, t, J = 4.4Hz), 3.79 - 3.76(1H, m), 3.68 - 3.64(3H, m), 3.56 - 3.52(4H, m), 3.50 - 3.48(2H, m), 3.43(1H, dd, J = 10.5, 6.5Hz), 3.37(1H, dd, J = 6.1Hz), 3.32(1H, dd, J = 8.5Hz), 3.29(1H, dd, J = 10.5, 3.1Hz), 3.22(3H, s), 3.12(1H, dd, J = 12.5, 5.8Hz), 2.93(1H, dd, J = 12.9, 6.5Hz), 2.49(2H, t, J = 1.7Hz). 13 C-NMR(150MHz, D2O - DMSO(d6) (1:40)) δ 166.4, 164.1, 160.2, 152.7, 149.8, 149.4, 148.0, 145.2, 140.7, 139.2, 133.4, 129.5, 127.8, 127.6, 124.9, 108.7, 107.3, 104.5, 104.5, 103.2, 99.8, 98.6, 82.8, 75.6, 73.3, 71.5, 71.3, 70.8, 70.5, 69.3, 68.2, 65.7, 62.2, 60.4, 58.4, 52.5, 52.4, 46.4, 42.4. ESI-TOF / MS(positive mode); Found: m / z 936.3410[(M + Na) + , Calcd. for C 39 H 51 N 11 O 15 Na: 936.3458.

[0141] tri-βGal4-1V209 (4-[2-(4-{[6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl]methyl}benzamido)acetamido]-N1,N7-bis[(1-{2-[(3-{(β-D-galacopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)amino}phenyl)amino]-2-oxoethyl}-1H-1,2,3-triazol-4-yl)methyl]-4-[3-({[1-(2-{(3-[(β-D-galacopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)amino]phenyl)amino}-2-oxoethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-3-oxopropyl]heptanediamide, Yield: 50%) 13 C-NMR (100 MHz, D2O-DMSO(d6)) δ. 1 H-NMR (600 MHz, D2O-DMSO(d6)(1:1)) δ 7.76 (3H, s), 7.58 (2H, d, J = 8.4 Hz), 7.23 (2H, d, J = 8.4 Hz), 6.95 (3H, dd, J = 8.4 Hz), 6.73 (3H, s), 6.65 (3H, d, J = 8.4 Hz), 6.36 (3H, d, J = 8.4 Hz), 5.12 (6H, s), 4.81 (2H, s), 4.27 (4H, d, J = 7.5 Hz), 4.20 (9H, d, J = 17.7 Hz), 3.81 (4H, s), 3.73 (2H, s), 3.68 (9H, s), 3.63 (9H, dd, J = 18.4, 2.7 Hz), 3.58 (4H, d, J = 10.2 Hz), 3.51 (7H, dd, J = 10.8, 4.1 Hz), 3.46 (8H, d, J = 6.1 Hz), 3.38 (5H, dd, J = 4.7 Hz), 3.36 (3H, d, J = 2.7 Hz), 3.32 (4H, dd, J = 8.5 Hz), 3.12 (2H, s), 2.91 (3H, dd, J = 10.2, 6.8 Hz). 1313C-NMR(150MHz, D2O-DMSO(d6)) δ 174.3, 164.3, 153.0, 148.9, 144.3, 137.8, 129.6, 127.5, 127.3, 127.3, 124.7, 108.9, 108.8, 104.2, 104.2, 102.8, 79.6, 74.7, 72.2, 70.8, 70.6, 70.1, 69.9, 68.0, 65.5, 61.5, 60.2, 57.8, 52.0, 45.7, 33.9, 29.3. ESI-TOF / MS (positive mode); Found: m / z 1176.9612 [(M + 2Na) 2+ , Calcd. for C 97 H 137 N 25 O 41 Na2: 2353.9188.

[0142] β-Glc6-1V209 (4 - ((6 - amino - 8 - hydroxy - 2 - (2 - methoxyethoxy) - 9H - purin - 9 - yl)methyl) - N - (β - D - glucopyranosyl) - (1→6) - (1 - deoxy - D - glucitol - 1 - yl)amino(phenyl) - 1H - 1,2,3 - triazol - 4 - yl)methyl)benzamide, Yield: 47%) 1 1H - NMR(400MHz, D2O(1 drop) - DMSO(d6)(1:40)) δ 7.92(1H, s), 7.76(2H, d, J = 8.2Hz), 7.33(2H, d, J = 8.2Hz), 7.00(1H, dd, J = 7.8, 8.2Hz), 6.83(1H, brs), 6.68(1H, d, J = 7.8Hz), 6.34(1H, d, J = 8.2Hz), 5.19(2H, s), 4.90(2H, s), 4.50(2H, s), 4.26 - 4.19(2H, m), 4.15(1H, d, J = 8.2Hz), 3.75 - 3.69(1H, m), 3.69 - 3.60(2H, m), 3.57 - 3.50(2H, m), 3.48 - 3.38(2H, m), 3.24 - 2.96(7H, m), 2.94 - 2.86(2H, m). 13C-NMR (100 MHz, D2O (1 drop) - DMSO (d6) (1:40)) δ 171.8, 167.3, 164.6, 160.7, 153.3, 150.0, 149.8, 145.6, 141.2, 139.4, 133.7, 130.1, 128.3, 128.2, 128.1, 125.5, 109.4, 108.2, 104.8, 104.0, 98.9, 77.1, 76.6, 74.1, 72.5, 72.4, 71.6, 70.8, 70.5, 70.5, 70.2, 66.2, 61.5, 58.8, 52.9, 46.4, 42.9, 35.4. ESI-TOF / MS (positive mode); Found: m / z 936.3457 [(M+Na) + , Calcd. for C 39 H 51 N 11 O 15 Na: 936.3458.

[0143] βGlc4-1V209 (4 - ((6 - amino - 8 - hydroxy - 2 - (2 - methoxyethoxy) - 9H - purin - 9 - yl)methyl) - N - (β - D - glucopyranosyl) - (1→4) - (1 - deoxy - D - glucitol - 1 - yl)amino(phenyl) - 1H - 1,2,3 - triazol - 4 - yl)methyl)benzamide, Yield: 45%) 1 H - NMR (400 MHz, D2O (1 drop) - DMSO (d6) (1:40)) δ 7.97 (1H, s), 7.82 (2H, d, J = 8.2 Hz), 7.39 (2H, d, J = 8.2 Hz), 7.04 (1H, dd, J = 7.8, 8.2 Hz), 6.90 (1H, brs), 6.74 (1H, d, J = 7.8 Hz), 6.41 (1H, d, J = 8.2 Hz), 5.25 (2H, s), 4.96 (2H, s), 4.56 (2H, s), 4.38 (1H, d, J = 6.9 Hz), 4.32 - 4.24 (2H, m), 3.89 - 3.81 (1H, m), 3.81 - 3.64 (4H, m), 3.64 - 3.52 (4H, m), 3.47 (1H, dd, J = 5.5, 11.0 Hz), 3.26 (3H, s), 3.25 - 2.96 (7H, m). ESI-TOF / MS (positive mode); Found: m / z 936.3457 [(M+Na) + , Calcd. for C 39 H 51 N 11 O 15 Na: 936.3458.

[0144] αGal6-1V209 (4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)-N-(α-D-galactopyranosyl)-(1→6)-(1-deoxy-D-glucitol-1-yl)amino(phenyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide, Yield: 36%) 1 H-NMR (400 MHz, D2O (1 drop)-DMSO (d6) (1:40)) δ 7.97 (1H, s), 7.82 (2H, d, J = 7.8 Hz), 7.39 (2H, d, J = 7.8 Hz), 7.05 (1H, dd, J = 7.3, 8.2 Hz), 6.88 (1H, brs), 6.73 (1H, d, J = 8.2 Hz), 6.39 (1H, d, J = 7.3 Hz), 5.24 (2H, s), 4.96 (2H, s), 4.71 (1H, d, J = 2.3 Hz), 4.55 (2H, s), 4.31 - 4.24 (2H, m), 3.82 - 3.66 (6H, m), 3.65 - 3.43 (8H, m), 3.26 (3H, s), 3.21 (1H, dd, J = 6.0, 12.8 Hz), 2.97 (1H, dd, J = 6.9, 12.8 Hz). 13 C-NMR (100 MHz, D2O (1 drop)-DMSO (d6) (1:40)) δ 167.3., 164.6, 160.7, 153.3, 150.0, 149.8, 145.6, 141.2, 139.4, 133.7, 130.2, 128.3, 128.1, 125.5, 108.2, 104.6, 103.9, 99.3, 98.9, 74.4, 72.4, 71.8, 70.8, 70.2, 70.0, 69.8, 69.5, 69.2, 66.2, 61.2, 58.8, 46.4, 42.9, 35.3. ESI-TOF / MS (positive mode); Found: m / z 936.3476 [(M+Na) + , Calcd. for C 39 H 51 N 11 O 15 Na: 936.3458.

[0145] αFuc2-βGal4-1V209(4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)-N-(α-D-fucosyl)-(1→2)-(β-D-glucopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)amino(phenyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide, Yield: 40%) 1 H-NMR (400 MHz, D2O (1 drop)-DMSO(d6) (1:40)) δ 7.93 (1H, s), 7.82 (2H, d, J = 7.8 Hz), 7.39 (2H, d, J = 7.8 Hz), 7.04 (1H, dd, J = 7.8, 7.8 Hz), 6.88 (1H, brs), 6.77 (1H, d, J = 7.8 Hz), 6.42 (1H, d, J = 7.8 Hz), 5.26 (2H, s), 5.09 (1H, s), 4.96 (2H, s), 4.55 (2H, s), 4.47 (1H, d, J = 6.8 Hz), 4.32 - 4.25 (2H, m), 3.91 - 3.83 (1H, m), 3.81 - 3.67 (3H, m), 3.67 - 3.40 (12H, m), 3.26 (3H, s), 3.26 - 3.18 (1H, m), 3.01 - 2.92 (1H, m), 1.07 (3H, d, J = 6.4). ESI-TOF / MS (positive mode); Found: m / z 000.0000 [(M+Na) + , Calcd. for C 45 H 61 N 11 O 19 Na: 1082.4037.

[0146] αXyl6-1V209 (4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)-N-(α-D-xylopyranosyl)-(1→6)-(1-deoxy-D-glucitol-1-yl)amino(phenyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide, Yield: 40%) 1 H-NMR(400MHz, D2O(1 drop)-DMSO(d6)(1:40)) δ 7.97(1H, s), 7.82(2H, d, J = 7.8Hz), 7.38(2H, d, J = 7.8Hz), 7.03(1H, dd, J = 7.8, 8.2Hz), 6.87(1H, brs), 6.75(1H, d, J = 1.8, 7.8Hz), 6.38(1H, d, J = 2.2, 8.2Hz), 5.24(2H, s), 4.94(2H, s), 4.63(1H, d, J = 3.2Hz), 4.55(2H, s), 4.47(1H, d, J = 6.8Hz), 4.32 - 4.25(2H, m), 3.91 - 3.83(1H, m), 3.81 - 3.67(3H, m), 3.67 - 3.40(12H, m), 3.26(3H, s), 3.26 - 3.18(1H, m), 3.01 - 2.92(1H, m), 1.07(3H, d, J = 6.4). ESI-TOF / MS(positive mode); Found: m / z 906.3458[(M + Na) + , Calcd. for C 38 H 49 N 11 O 14 Na: 906.3353.

[0147] αAra5-1V209 (4-((6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl)-N-(α-D-arabinopyranosyl)-(1→5)-(1-deoxy-D-arabinitol-1-yl)amino(phenyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide, Yield: 16%) ESI-TOF / MS (positive mode); Found: m / z 876.3247 [(M+Na) + , Calcd. for C 37 H 47 N 11 O 13 Na: 876.3247.

[0148] αMan6-1V209 (4 - [{6 - amino - 8 - hydroxy - 2 - (2 - methoxyethoxy) - 9H - purin - 9 - yl}methyl] - N - [(1 - {2 - ([3 - {(α - D - mannopyranosyl)-(1→6)-(1 - deoxy - D - glucitol - 1 - yl)-amino}phenyl]amino] - 2 - oxoethyl}-1H - 1,2,3 - triazol - 4 - yl}methyl]benzamide, Yield: 48%) 1 H - NMR (600 MHz, D2O - DMSO(d6)(1:40)) δ 7.97 (1H, s), 7.82 (2H, d, J = 8.2 Hz), 7.38 (2H, d, J = 8.2 Hz), 7.02 (1H, dd, J = 8.2 Hz), 6.87 (1H, s), 6.74 (1H, d, J = 8.2 Hz), 6.37 (1H, d, J = 8.2 Hz), 5.24 (2H, s), 4.94 (2H, s), 4.64 (1H, d, J = 4.1 Hz), 4.54 (2H, s), 4.20 (2H, d, J = 4.1 Hz), 3.78 (1H, s), 3.69 (1H, t, J = 5.4 Hz) 3.64 - 3.34 (18H, m), 3.22 - 3.17 (4H, m), 3.12 (1H, dd, J = 12.9 Hz), 2.88 (1H, dd, J = 12.6, 4.6 Hz), 2.04 (2H, s). 1313C-NMR: (150 MHz, D2O - DMSO(d6) (1:40)) δ 166.4, 165.8, 163.6, 152.9, 149.1, 148.4, 145.0, 144.6, 140.5, 139.0, 133.3, 129.1, 127.5, 127.3, 124.6, 108.8, 108.4, 106.8, 106.6, 102.7, 102.4, 99.5, 99.3, 73.0, 72.7, 72.5, 70.8, 70.5, 70.2, 69.5, 68.7, 68.6, 66.5, 66.1, 60.5, 57.9, 51.2, 46.4, 42.1. ESI - TOF / MS (positive mode); Found: m / z 936.3471 [(M + Na) + , Calcd. for C 39 H 51 N 11 O 15 Na: 936.3458.

[0149] (βGlcNAc4)4 - 1V209 (4 - [{6 - amino - 8 - hydroxy - 2 - (2 - methoxyethoxy) - 9H - purin - 9 - yl}methyl] - N - [(1 - {2 - [(3 - {(2 - acetamido - 2 - deoxy - β - D - glucopyranosyl)-(1→4)-(2 - acetamido - 2 - deoxy - β - D - glucopyranosyl)-(1→4)-(2 - acetamido - 2 - deoxy - β - D - glucopyranosyl)-(1→4)-(2 - acetamido - 2 - deoxy - β - D - glucopyranosyl)-(1→4)-(2 - acetamido - 1,2 - deoxy - D - glucitol - 1 - yl) - amino}phenyl)amino] - 2 - oxoethyl} - 1H - 1,2,3 - triazol - 4 - yl)methyl]benzamide, Yield: 38%) ESI - TOF / MS (positive mode); Found: m / z 1627.6375 [(M + Na) + , Calcd. for C 67 H 96 N 16 O 30Na: 1627.6370.

[0150] (βGlc3)4-1V209(4-[{6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl}methyl]-N-[(1-{2-[(3-{(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(1-deoxy-D-glucitol-1-yl)-amino}phenyl)amino]-2-oxoethyl}-1H-1,2,3-triazol-4-yl)methyl]benzamide, Yield: 84%) 1 1H-NMR (600 MHz, D2O-DMSO(d6)(1:40)) δ 7.85(1H, s), 7.65(3H, d, J = 8.2 Hz), 7.29(4H, d, J = 8.2 Hz), 6.98(1H, dd, J = 8.2 Hz), 6.75(1H, s), 6.64(1H, d, J = 8.2 Hz), 6.37(1H, d, J = 8.2 Hz), 5.14(2H, s), 4.87(4H, s), 4.51 - 4.43(6H, m), 4.42 - 4.40(5H, m), 4.36 - 4.34(1H, m), 4.21 - 4.17(4H, m), 3.93 - 3.87(2H, m), 3.81 - 3.78(1H, m), 3.69 - 3.60(7H, m), 3.59 - 3.55(1H, m), 3.52 - 3.29(19H, m), 3.29 - 3.13(9H, m), 3.06 - 3.04(3H, m), 2.95 - 2.93(1H, m), 2.51(2H, d, J = 3.6 Hz). ESI-TOF / MS (positive mode) Found: m / z 1422.5053[(M + Na) + , Calcd. for C 57 H 81 N 11 O 30 Na: 1422.5043.

[0151] tri-αMan6-1V209 (4-[2-(4-{[6-Amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl]methyl}benzamido)acetamido]-N1,N7-bis[(1-{2-[(3-{(α-D-mannopyranosyl)-(α1→6)-(1-deoxy-D-glucitol-1-yl)amino}phenyl)amino]-2-oxoethyl}-1H-1,2,3-triazol-4-yl)methyl]-4-[3-({[1-(2-{(3-[(α-D-mannopyranosyl)-(1→6)-(1-deoxy-D-glucitol-1-yl)amino]phenyl)amino}-2-oxoethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-3-oxopropyl]heptanediamide, Yield: 30%) 1 1H-NMR (600 MHz, D2O-DMSO(d6)(1:1)) δ 7.75 (3H, s), 7.58 (2H, d, J = 8.4 Hz), 7.23 (1H, d, J = 8.4 Hz), 6.96 (2H, dd, J = 8.4 Hz), 6.73 (3H, d, J = 8.4 Hz), 6.64 (3H, d, J = 8.4 Hz), 6.35 (3H, d, J = 8.4 Hz), 5.11 (6H, s), 4.83 (2H, d, J = 10.9 Hz), 4.61 (4H, s), 4.21 (8H, d, J = 10.4 Hz), 3.72 (6H, s), 3.68 (5H, s), 3.64 (6H, dd, J = 7.5 Hz), 3.61 (9H, s), 3.57 (5H, dd, J = 10.2, 2.7 Hz), 3.51 (7H, dd, J = 11.6, 4.8 Hz), 3.46 (9H, dd, J = 8.5 Hz), 3.43 (6H, s), 3.40 (3H, d, J = 10.2 Hz), 3.12 (4H, dd, J = 10.2, 4.8 Hz), 2.91 (3H, dd, J = 10.2, 8.1 Hz). ESI-TOF / MS (positive mode); Found: m / z 1176.9563 [(M + 2Na) 2+ , Calcd. for C 97 H 137 N 25 O41 Na2: 2353.9188.

[0152] tri-αGlc4-1V209 (4-[2-(4-{[6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl]methyl}benzamido)acetamido]-N1,N7-bis[(1-{2-[(3-{(α-D-glucopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)amino}phenyl)amino]-2-oxoethyl}-1H-1,2,3-triazol-4-yl)methyl]-4-[3-({[1-(2-{(3-[(α-D-glucopyranosyl)-(1→4)-(1-deoxy-D-glucitol-1-yl)amino]phenyl)amino}-2-oxoethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-3-oxopropyl]heptanediamide, Yield: 28%) 1 H-NMR (600 MHz, D2O-DMSO(d6) (1:1)) δ 7.73 (3H, s), 7.55 (2H, d, J = 8.4 Hz), 7.21 (2H, d, J = 8.4 Hz), 6.94 (3H, dd, J = 8.4 Hz), 6.71 (3H, s), 6.60 (3H, d, J = 8.4 Hz), 6.33 (3H, d, J = 8.4 Hz), 5.10 (6H, s), 4.85 (3H, d, J = 3.4 Hz), 4.79 (2H, s), 4.23 - 4.15 (9H, m), 3.71 - 3.67 (10H, m), 3.67 - 3.64 (8H, m), 3.63 (3H, dd, J = 5.4, 2.0 Hz), 3.62 - 3.58 (6H, m), 3.52 - 3.45 (14H, m), 3.39 (4H, dd, J = 11.6, 6.8 Hz), 3.31 (4H, dd, J = 10.2, 5.4 Hz), 3.17 (3H, d, J = 9.6 Hz), 3.14 (3H, d, J = 3.4 Hz), 3.08 (3H, dd, J = 12.9, 4.0 Hz), 2.97 (3H, dd, J = 12.9, 7.4 Hz). 13C-NMR(150MHz, D2O-DMSO(d6)(1:1)) δ 176.2, 166.1, 154.9, 150.4, 146.1, 139.4, 131.3, 129.0, 126.4, 111.4, 110.6, 106.6, 106.0, 102.0, 101.9, 100.0, 84.9, 83.8, 74.3, 73.9, 73.7, 72.5, 71.6, 70.7, 70.4, 70.3, 65.4, 63.5, 61.8, 59.5, 59.4, 53.7, 47.7, 35.7, 31.0. ESI-TOF / MS(positive mode); Found: m / z 1176.9616 [(M + 2Na) 2+ , Calcd. for C 97 H 137 N 25 O 41 Na2: 2353.9188.

[0153] tri-(βGlc3)4-1V209(4-[2-(4-{(6-amino-8-hydroxy-2-(2-methoxyethoxy)-9H-purin-9-yl)methyl}benzamido)acetamido]-N1,N7-bis[(1-{2-[(3-{(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(1-deoxy-D-glucitol-1-yl)amino}phenyl)amino]-2-oxoethyl}-1H-1,2,3-triazol-4-yl)methyl]-4-[3-({[1-(2-{(3-[(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(β-D-glucopyranosyl)-(1→3)-(1-deoxy-D-glucitol-1-yl)amino]phenyl)amino}-2-oxoethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-3-oxopropyl]heptanediamide, Yield: 29%) 1 H-NMR(600MHz,D2O-DMSO(d6)(1:1))δ7.75(3H,s),7.57(2H,d,J=8.4Hz),7.23(2H,d,J=8.4Hz),6.96(3H,dd,J=8.4Hz),6.72(3H,s),6.65(3H,d,J=8.4Hz),6.37(3H,d,J=8.4Hz),5.11(6H,s),4.56-4.43(13H,m),4.25-4.18(10H,m),3.92(3H,brs),3.83-3.80(4H,m),3.72-3.65(19H,m),3.64-3.58(5H,m),3.52-3.35(48H,m),3.33-3.29(10H,m),3.27-3.16(30H,m),3.15-3.08(14H,m). ESI-TOF / MS(positive mode);Found:m / z 1897.7229[(M+Na) + ],Calcd.for C 160 H 245 N 25 O 77 Na:1897.7904.

[0154] (αGlc6) n -1V209(MW17,000) 1 H-NMR(400MHz,D2O)δ7.83(1H,br),7.46(2H,br),7.11(2H,br),6.94(1H,br),6.69(1H,br),6.56(1H,br),6.39(1H,br),4.96-4.76(~100H,m),4.01-3.03(~600H,m).

[0155] (αGlc6) n -1V209(MW40,000) 1 H-NMR(400MHz,D2O)δ7.88(1H,br),7.56(2H,br),7.21(2H,br),7.04(1H,br),6.78(1H,br),6.65(1H,br),6.48(1H,br),4.90-4.77(~160H,m),4.03-3.08(~960H,m).

[0156] [Test Example 1: Measurement of immune enhancing activity of glycan-TLR ligand complex] The immune-enhancing activity of the glycan-TLR ligand complex prepared in Production Example 5 was evaluated. In the evaluation of immune-enhancing activity, the amount of cytokine (IL-6) production induced by the glycan-TLR ligand complex in mouse bone marrow derived dendritic cells (hereinafter referred to as "mBMDC") was quantified by ELISA. First, 1×10 cells per well were placed in a 96-well plate. 5 150 μL of the cell suspension was seeded onto each well and incubated at 37° C. in 5% CO 2 for 18 hours.

[0157] Next, the concentration of the glycan-TLR ligand complex was adjusted to 8 μM. The resulting glycan-TLR ligand complex solution was serially diluted 4-fold, and 50 μL of the solution was added to each well of the 96-well plate and incubated at 37°C and 5% CO2 for 18 hours. Here, (i) a solution of the TLR ligand (1V209) not complexed with a glycan (8 μM) serially diluted 4-fold was also added to each well of the 96-well plate and incubated at 37°C and 5% CO2 for 18 hours.

[0158] Next, ELISA was performed according to the following procedure. First, 50 μL of anti-mouse IL-6 antibody diluted 250-fold with Coating Buffer was added to a 96-well ELISA plate and incubated at 4°C for 18 hours. After removing the antibody solution, each well was washed three times with 300 μL of PBS solution containing 1% Tween 20 (hereinafter referred to as "1% PBS / T"). Then, 300 μL of 1% bovine serum albumin-PBS solution (hereinafter referred to as "blocking buffer") was added to the well and incubated for 2 hours under conditions of 37°C and 5% CO2. After removing the blocking buffer, each well was washed three times with 300 μL of 1% PBS / T.

[0159] Next, 150 μL of the culture supernatant was transferred to another 96-well plate, and 50 μL of it was transferred to an ELISA plate on which anti-IL-6 antibody was immobilized. After incubating the ELISA plate at 4° C. for 2 hours, the culture supernatant was removed, and each well was washed three times with PBS containing 0.05% Tween 20 (hereinafter referred to as “0.05% PBS / T”). Then, 50 μL of biotin-labeled anti-IL-6 antibody diluted 1000-fold with blocking buffer was added to each well, and incubated at room temperature for 2 hours. After removing the antibody solution, each well was washed three times with 300 μL of 1% PBS / T, and 50 μL of HRP-modified streptavidin solution diluted 1000-fold with blocking buffer was added. After incubation at room temperature for 1 hour, each well was washed five times with 1% PBS / T. Then, 50 μL of TMB Microwell Peroxidase Substrate (manufactured by Nacalai Tesque) was added, and chemiluminescence was observed. Then, 50 μL of 1M o-Phosphoric Acid solution was added to stop the chemiluminescence. Then, the ELISA plate was immediately placed on a plate reader and the absorbance at 450 nm was measured. The amount of IL-6 produced in the culture supernatant was analyzed using GraphPad Prism 7 software. In addition, ELISA was performed in the same manner using recombinant mouse IL-6 (manufactured by eBioscience) with a known concentration, and a calibration curve was created.

[0160] (result) Figure 1 shows the amount of IL-6 produced by adding a solution of a glycan-TLR ligand complex to mBMDCs. The amount of TLR7 ligand (μM) shown in Figure 1 was calculated from the concentration of the glycan-TLR ligand complex added, and indicates the amount actually added. It was confirmed that the TLR ligand complex increased IL-6 production in a TLR7 ligand concentration-dependent manner.

[0161] Table 1 shows the 50% effective concentration (EC) obtained from the amount of IL-6 produced by adding the glycan-TLR ligand complex solution to mBMDC. 50 ) is shown. EC 50 The lower the value, the stronger the immune enhancing effect. It was confirmed that the immune enhancing effect of the TLR ligand complex was higher than that of 1V209.

[0162] [Table 1]

[0163] [Test Example 2: Measurement of immune enhancing activity using chicken-derived OVA and glycan TLR ligand complex] The in vivo immune enhancing activity of the sugar chain TLR ligand complex (3NH-αCD-1V209, formula (24)) shown below, prepared by the method described in Non-Patent Document 3, was evaluated using BALB / cA mice (7 weeks old, female). OVA was used as a model antigen, and a mixed solution (20 μL) of OVA (20 μg) and 3NH-αCD-1V209 (2 nmol or 20 nmol) was intranasally administered to mice three times on days 0, 7, and 14 (4 or 8 mice per group). 1V209 (2 nmol) and Cholera toxin (5 μg) were used as controls.

[0164] [ka]

[0165] On the 21st day after the start of immunization, blood, nasal wash, and spleen were collected. Blood was collected from the orbital venous plexus under sevoflurane inhalation anesthesia. The collected blood was centrifuged (4,000×g, 10 min, 4°C) and the serum layer was collected. Nasal wash was collected by injecting Otsuka distilled water (300 μL) near the esophagus with a syringe (27G×3 / 4'). The serum layer was centrifuged again (4,000×g, 10 min, 4°C) and the supernatant was collected. IgA and IgG antibodies in the nasal wash and serum were evaluated by ELISA.

[0166] In the ELISA, first, a protein solution diluted with PBS (OVA: 5 μg / mL, 50 μL / well) was added to each well of a 96-well ELISA plate and incubated at 4°C for 18 hours. After removing the protein solution, each well was washed three times with a PBS solution containing 0.05% Tween 20 (hereinafter referred to as "PBS / T", 300 μL / well). Then, a blocking buffer (300 μL / well) was added and incubated on a plate shaker (400 rpm, room temperature) for 2 hours. After removing the blocking buffer, each well was washed three times with PBS / T (300 μL / well). Next, a serum solution or nasal wash (50 μL / well) diluted with the blocking buffer was added and incubated on a plate shaker (400 rpm, room temperature) for 2 hours. In this case, in the case of serum, a serum solution (50 μL / well) diluted 25-fold with blocking buffer was added to the first dose, and a solution serially diluted 4-fold (50 μL / well) was added to the second to eighth doses. In the case of nasal wash, a nasal wash (50 μL / well) diluted 2-fold with blocking buffer was added to the first dose, and a solution serially diluted 2-fold (50 μL / well) was added to the second to eighth doses. After removing the serum solution or nasal wash, the plate was washed five times with PBS / T, and goat anti-mouse IgAα chain (HRP) diluted 10,000-fold with blocking buffer or anti-IgG (H+L chain) (mouse) pAb-HRP (50 μL / well) diluted 5,000-fold with blocking buffer was added, and the plate was incubated for 1.5 hours on a plate shaker (400 rpm, room temperature). After removing the antibody solution, the plate was washed five times with PBS / T. Then, TMB Microwell Peroxidase Substrate (50 μL / well) was added, and chemiluminescence was observed. The reaction was stopped by adding o-Phosphoric Acid solution (1 M, 50 μL / well). The absorbance at 450 nm was then measured immediately using a plate reader. GraphPad Prism7 was used for statistical analysis.

[0167] (result) Figures 2(A) and 2(B) show the IgA antibody production titers against OVA in nasal washings and serum, respectively. Figures 2(C) and 2(D) show the IgG antibody production titers against OVA in nasal washings and serum, respectively. It was shown that in the group administered the 3NH-αCD-1V209 mixture, the IgA and IgG antibody production ability against OVA was improved by 10 to 100 times.

[0168] There were no significant differences in body weight between groups, and as shown in FIG. 3, there were no significant differences in spleen weight between groups.

[0169] [Test Example 3: Measurement of immune enhancing activity using influenza virus-derived HA and glycan TLR ligand complex (3NH-αCD-1V209)] Recombinant HA (A / Pueruto Rico / 8 / 1934, hereinafter also referred to as "Flu HA"), which is a spike protein antigen of influenza virus, was used to evaluate immune enhancing activity in the same manner as in Test Example 2 (6 mice per group). In the evaluation of immune enhancing activity, a mixture (20 μL) of Flu HA (5 μg) and 3NH-αCD-1V209 (20 nmol) was administered intranasally according to the same immunization schedule as in Test Example 2. On the 21st day after the start of immunization, blood and nasal washes were collected. IgA and IgG antibodies in the nasal washes and serum were quantified by ELISA in the same manner as in Test Example 2, except that a protein solution containing 5 μg / mL Flu HA was used instead of 5 μg / mL OVA.

[0170] (result) Figures 4(A) and 4(B) show the IgA antibody production titers against HA in nasal washings and serum, respectively. Figures 4(C) and 4(D) show the IgG antibody production titers against Flu HA in nasal washings and serum, respectively. In the group administered a mixture of HA and 3NH-αCD-1V209, it was shown that the antibody titers of IgA and IgG antibodies were 10 to 1000 times higher in both nasal washings and serum than in the group administered Flu HA alone.

[0171] [Test Example 4: Measurement of immune enhancing activity using influenza vaccine and glycan TLR ligand complex (3NH-αCD-1V209)] A commercially available influenza vaccine (Denka Seiken, two types of type A and two types of type B, hereafter also referred to as "Flu vaccine") was used to evaluate immune enhancing activity in the same manner as in Test Example 3 (six mice per group). A mixed solution (50 μL) of Flu vaccine (total HA amount approximately 5 μg) and 3NH-αCD-1V209 (20 nmol) was administered intranasally according to the same immunization schedule as in Test Example 3. On the 21st day after the start of immunization, blood and nasal washes were collected. IgA and IgG antibodies in the nasal washes and serum were evaluated by ELISA in the same manner as in Test Example 3.

[0172] Flu vaccine (more than 30μg per strain of influenza virus HA fraction in 1ml, 40μL) was mixed with PBS (10μL) or 3NH-αCD-1V209 (20nmol, 2mM, 10μL) and intranasally administered to BALB / cA mice (7 weeks old, female) on days 0, 7, and 14. Blood and nasal washes were collected on day 21 after the start of immunization. IgA and IgG antibodies in the nasal washes and serum were quantified by ELISA in the same manner as in Test Example 3.

[0173] (result) Figures 5(A) and 5(B) show the IgA antibody titers against HA in nasal washings and serum, respectively. Figures 5(C) and 5(D) show the IgG antibody titers against HA in nasal washings and serum, respectively. In the group administered the mixture of Flu vaccine and 3NH-αCD-1V209, the antibody titers of IgA and IgG antibodies were shown to be 5-30 times higher than in the group administered Flu vaccine alone.

[0174] [Test Example 5: Evaluation of virus neutralizing activity of antibodies produced in the nasal mucosa] In order to evaluate whether the virus infection can be inhibited, a neutralizing activity test was performed using influenza virus type B (field isolate) using the nasal wash collected in Test Example 4. MDCK cell line was used as the cells. Influenza virus type B was mixed with the nasal wash, added to the cell solution, and cultured for 4 days. The cells were stained with crystal violet to evaluate the infection inhibition effect. With crystal violet staining, the cells are stained when the virus infection is inhibited and the cells are alive, but are not stained when the cells are killed by the virus infection, so the presence or absence of virus infection can be evaluated.

[0175] MDCK cells (3 × 10 4 Then, 120 TCID 50 The cells were mixed with nasal wash (60 μL, serially diluted 10-fold from the original solution with E-MEM infection medium) and incubated for 1 hour at 34°C and 5% CO2. The cell culture medium was removed, and the cells were washed twice with PBS and once with E-MEM infection medium. After that, a mixed solution of nasal wash (50 μL) and virus solution (50 μL) (the sample incubated for 1 hour above) was added and cultured for 4 days at 34°C and 5% CO2. The culture supernatant was then removed and the cells were washed with PBS. The cells were fixed with 70% ethanol (5 minutes), and after removing the 70% ethanol, 0.4% crystal violet solution (100 μL) was added and incubated at room temperature for 30 minutes, after which the crystal violet was removed and the cells were washed with water.

[0176] (result) As shown in Figure 6, in the group administered Flu vaccine alone, antibodies with neutralizing activity were induced only in some mice. On the other hand, in the group administered a mixture of 3NH-αCD-1V209 and Flu vaccine, production of antibodies with neutralizing activity was induced in all mice. Therefore, it was demonstrated that 3NH-αCD-1V209 enhances the production of antibodies with infection inhibitory activity against influenza virus.

[0177] [Test Example 6: Influenza virus infection protection test using mice] The protective effect against influenza virus infection was evaluated using BALB / cA mice (7 weeks old, female, 5 mice per group) with the influenza A H1N1 Puerto Rico strain (A / Pueruto Rico / 8 / 1934).

[0178] The animals were immunized with Flu HA in the same manner as in Test Example 3, and with Flu vaccine in the same manner as in Test Example 4. Seven days after the final immunization, they were administered a lethal dose of influenza virus (50 μL, 25 μL in each nostril, 1 × 10 4 TCID 50 Mice were intranasally infected with 100 μL of 1000 μl ...

[0179] (result) Figure 7(A) and Figure 7(B) show the time course of body weight change and survival rate after infection, respectively. In the groups administered 3NH-αCD-1V209 (3NH-αCD-1V209+Flu HA and 3NH-αCD-1V209+Flu vaccine), there was almost no weight loss and no death was confirmed. On the other hand, in the groups not administered 3NH-αCD-1V209 (PBS, PBS+Flu HA and PBS+Flu vaccine), there was weight loss and all animals died. These results demonstrated that the glycan-TLR ligand complex is useful as an adjuvant for mucosal vaccines.

[0180] [Test Example 7: Influenza virus infection protection test using mice] Using BALB / cA mice (7-week-old, female, 5 mice per group), the in vivo immune enhancing activity of tri-βGal4-1V209 (above formula (12)) obtained in Production Example 5 was evaluated for its protective effect against influenza virus infection. The influenza virus used was the type A H1N1 Puerto Rico strain (A / Pueruto Rico / 8 / 1934).

[0181] A solution (20 μL) of a mixture of influenza virus spike protein antigen recombinant HA (A / Pueruto Rico / 8 / 1934, hereafter also referred to as "Flu HA", 5 μg) and tri-βGal4-1V209 (20 nmol) was intranasally administered twice to mice. 22 days after the final immunization, a lethal dose of influenza virus (50 μL, 25 μL in each nostril, 1 × 10 4 TCID 50 Mice were intranasally infected with 100 μL of 1000 μl ...

[0182] (result) Figure 8(A) and Figure 8(B) show the time course of body weight change and survival rate after infection, respectively. In the group administered tri-βGal4-1V209 (tri-βGal4-1V209+Flu HA), there was almost no weight loss and no deaths were observed. On the other hand, in the groups not administered tri-βGal4-1V209 (PBS and PBS+Flu HA), there was a loss of body weight and all animals died. These results demonstrated that the glycan-TLR ligand complex is useful as a vaccine adjuvant.

[0183] The above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not by the embodiment. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0184] The present invention is useful for vaccine compositions, particularly mucosal vaccine compositions.

Claims

1. a plurality of first ligand moieties that bind to a C-type lectin receptor; a second ligand moiety that binds to a Toll-like receptor; a linker having a branched structure in which the plurality of first ligand moieties and the second ligand moiety are each bonded to an end thereof; A compound having the formula:

2. Formula (1) 【Chemistry 1】 (In formula (1), R Ｃ has the first ligand moiety, and R Ｔ has the second ligand portion, and p １ ~p ３ are each independently an integer of 0 to 6. having the structure shown in The compound of claim 1.

3. The structure of the first ligand portion is A structure in which a monosaccharide selected from the group consisting of glucose, galactose, mannose, amino sugars and acidic sugars is bound to the linker; or a structure in which one or more oligosaccharides or polysaccharides selected from the group consisting of glucose, galactose, mannose, amino sugars and acidic sugars are bound to the linker; 3. A compound according to claim 1 or 2.

4. The second ligand moiety is Binds to at least one selected from the group consisting of Toll-like receptor 4, Toll-like receptor 7, and Toll-like receptor 8; 3. A compound according to claim 1 or 2.

5. The structure of the second ligand portion is 1V209 is bound to the linker; 3. A compound according to claim 1 or 2.

6. a plurality of first ligand moieties that bind to a C-type lectin receptor; a second ligand moiety that binds to a Toll-like receptor; a linker having a branched structure in which the plurality of first ligand moieties and the second ligand moiety are each bonded to an end thereof; Compounds having the formula: Immune enhancer.

7. A vaccine adjuvant, The immune enhancing agent according to claim 6.

8. It is a mucosal immune enhancer. The immune enhancing agent according to claim 6.

9. An adjuvant for mucosal vaccines, The immune enhancing agent according to claim 8.

10. The immune enhancing agent according to claim 7 , An antigen; 23. A vaccine composition comprising:

11. The immune enhancing agent according to claim 9 , An antigen; A mucosal vaccine composition comprising:

12. For administration to the nasal mucosa, The mucosal vaccine composition according to claim 11.

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