Immunopotentiator containing a polynucleotide-peptide conjugate and pharmaceutical composition containing the same

Abstract

The present invention provides an immune inducer containing a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof as an active ingredient. The polynucleotide-peptide conjugate comprises a single-stranded polynucleotide or polynucleotide derivative including CpG motifs, a peptide, and a spacer covalently bonded to the polynucleotide or polynucleotide derivative at one end and covalently bonded to the peptide at the other end. In the peptide, one or more contiguous amino acids at the N terminal of an MHC binding peptide are replaced by amino acids having a reactive functional group for covalently binding to the spacer. The one or more contiguous amino acids do not include an anchor residue for forming an MHC bond.

Description

Technical Field

[0001] The present invention relates to an immune inducer containing a polynucleotide-peptide conjugate as an active ingredient, a pharmaceutical composition containing the same, and the like.

Background Art

[0002] The Toll-like receptor (TLR) family present in antigen-presenting cells such as dendritic cells, macrophages, and B cells reacts with various pathogens, induces the production of cytokines, and promotes the differentiation of naive T cells into Th1 cells and the activation of killer T cells, etc., to induce acquired immunity. The components of pathogens recognized by a series of TLR families are diverse, and one of them is DNA having a CpG motif (CpG DNA), which is a ligand for TLR9. The CpG motif is based on six bases with cytosine (C) and guanine (G) arranged in the center and two purine bases and two pyrimidine bases arranged in front and behind, and is represented by the sequence -PuPu-CG-PyPy- (Pu represents a purine base and Py represents a pyrimidine base. In the case of humans, it is known that GTCGTT also has ligand activity for TLR9), and it is a base sequence that is rare in mammals and abundant in microorganisms. In mammals, most of the few existing CpG motifs are methylated. The unmethylated CpG motif, which hardly exists in mammals, has strong immunostimulatory activity (see, for example, Non-Patent Documents 1 to 3). CpG DNA taken into cells by endocytosis is recognized by TLR9 present in phagosome-like endoplasmic reticulum, resulting in the production of Th1 cytokines such as interferon-γ (IFN-γ) and interleukin-2 (IL-2), and strongly inducing a Th1 response. The Th1 response suppresses allergic reactions in which the Th2 response is dominant, and activates macrophages and cytotoxic T cells (CTLs) to have strong antitumor activity by cellular immunity.

[0003] CpG DNA is expected to be an adjuvant not only for preventing infection but also for allergic diseases and neoplastic diseases. For example, the following reports are available on conjugates in which CpG DNA is covalently bound to an antigen. Non-Patent Document 4 describes that a conjugate of CpG DNA and an 18-24 mer peptide derived from ovalbumin (OVA) antigen promoted uptake into dendritic cells and antigen presentation. Non-Patent Documents 5 and 6 describe that a conjugate of CpG DNA and OVA antigen protein induces T cell activation in vitro. Non-Patent Document 5 also describes that antigen-specific cytotoxic activity was induced in vivo. Non-Patent Document 7 describes a method for preparing a conjugate of a CpG oligonucleotide and a tumor-related protein or cell. Patent Document 1 discloses an immunopotentiator containing, as an active ingredient, a conjugate of a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif and an antigenic peptide, and it has been shown to have high antigen-specific immunopotentiating activity.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0006] In Patent Document 1, the inventors showed that CpG DNA-peptide conjugates have high cytotoxic T lymphocyte (CTL) induction ability through research using peptides having antigenicity derived from OVA. On the other hand, it was found that when similar CpG DNA-peptide conjugates were prepared for other antigen peptides, sufficient CTL induction ability might not be obtained. An object of the present invention is to provide an immunopotentiator capable of inducing CTL activity for a wider range of antigen peptides and the like.

Means for Solving the Problems

[0007] As a result of studying peptides that do not have sufficient CTL induction ability when made into CpG DNA-peptide conjugates, the inventors found that when an amino acid such as cysteine is added to the N-terminus of the peptide for conjugation, the peptide that was originally bindable to MHC molecules (MHC-binding peptide) becomes unable to bind to MHC molecules. Furthermore, by using a peptide in which one or more consecutive amino acids at the N-terminus that do not contain an anchor residue are replaced with an amino acid such as cysteine, rather than a peptide in which an amino acid such as cysteine is directly added to the N-terminus of the MHC-binding peptide, it was found that a CpG DNA-peptide conjugate having high CTL induction ability can be prepared. The inventors further conducted intensive research and completed the present invention.

[0008] That is, the present invention includes the following inventions. [1] An immunomodulator containing a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof as an active ingredient, The polynucleotide-peptide conjugate comprises a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, a peptide, and a spacer that is covalently bonded to the polynucleotide or polynucleotide derivative at one end and covalently bonded to the peptide at the other end, The peptide is a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced with amino acids having a reactive functional group for forming a covalent bond with the spacer, where the one or more consecutive amino acids do not include an anchor residue for MHC binding, the immunomodulator. [2] The immunomodulator according to [1], wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide are covalent bonds cleavable in a biological environment. [3] The immunomodulator according to [1] or [2], wherein the amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an analog thereof having a thiol group. [4] The immunomodulator according to any one of [1] to [3], wherein the covalent bond between the spacer and the peptide is a disulfide bond. [5] The immunomodulator according to any one of [1] to [4], wherein the MHC-binding peptide is an MHC-I-binding peptide. [6] The immunomodulator according to [5], wherein the MHC-I-binding peptide is an HLA-A-binding peptide or an HLA-B-binding peptide. [7] The immunomodulator according to [5] or [6], wherein the amino acid length of the MHC-I-binding peptide is 8 or more and 11 or less. [8] The immunomodulator according to any one of [1] to [4], wherein the MHC-binding peptide is an MHC-II-binding peptide. [9] The immunopotentiator according to any one of [1] to [8], wherein the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs.

[10] The immunopotentiator according to any one of [1] to [9], wherein the base length of the polynucleotide or polynucleotide derivative is 15 or more and 40 or less.

[11] The immunopotentiator according to

[10] , wherein the base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less.

[12] The immunopotentiator according to any one of [1] to

[11] , wherein the polynucleotide or polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bonds is replaced with phosphorothioate bonds.

[13] The immunopotentiator according to

[12] , wherein in the polynucleotide derivative in which at least a part of the phosphodiester bonds is replaced with phosphorothioate bonds, 50% or more of the phosphodiester bonds are replaced with phosphorothioate bonds.

[14] The immunopotentiator according to

[13] , wherein in the polynucleotide derivative in which at least a part of the phosphodiester bonds is replaced with phosphorothioate bonds, 90% or more of the phosphodiester bonds are replaced with phosphorothioate bonds.

[15] The immunopotentiator according to any one of [1] to

[14] , wherein the spacer contains a repeating unit represented by the following formula.

Chemical formula

[16] The immunopotentiator according to any one of [1] to

[15] , wherein the spacer has a structure represented by any of the following formulas.

Chemical formula

[17] The immunopotentiator according to any one of [1] to

[14] , wherein the spacer has a structure represented by any of the following formulas.

Chemical formula

[18] An immunopotentiator containing, as an active ingredient, a polynucleotide-peptide conjugate described in [1] or a pharmaceutically acceptable salt thereof, wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide is a covalent bond cleavable in a biological environment, wherein the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs, wherein the polynucleotide or polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bond is replaced with a phosphorothioate bond, said immunopotentiator.

[19] An immunopotentiator containing, as an active ingredient, a polynucleotide-peptide conjugate described in [1] or a pharmaceutically acceptable salt thereof, wherein the amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an analog thereof having a thiol group, wherein the covalent bond between the spacer and the peptide is a disulfide bond, wherein the MHC-binding peptide is an MHC-I-binding peptide, wherein the MHC-I-binding peptide is an HLA-A-binding peptide or an HLA-B-binding peptide, wherein the amino acid length of the MHC-I-binding peptide is 8 or more and 11 or less, The polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs, the base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less, in the polynucleotide derivative in which at least a part of the phosphodiester bond is replaced with a phosphorothioate bond, 90% or more of the phosphodiester bond is replaced with a phosphorothioate bond, the spacer has a structure represented by any of the following formulas, the immune inducer. [Chemical formula]

[20] The immune inducer according to any one of [1] to

[19] , further comprising a substance having an immune activation activity as an adjuvant.

[21] A pharmaceutical composition comprising the immune inducer according to any one of [1] to

[20] .

[22] The pharmaceutical composition according to

[21] , which is for the treatment or prevention of infectious diseases, tumors, or allergic diseases.

[23] The pharmaceutical composition according to

[21] , which is for the treatment or prevention of tumors.

[24] A method for treating or preventing infectious diseases, tumors, or allergic diseases, comprising administering to a patient the immune inducer according to any one of [1] to

[20] .

[25] A method for treating or preventing tumors, comprising administering to a patient the immune inducer according to any one of [1] to

[20] .

[26] The immune inducer according to any one of [1] to

[20] for use in the treatment or prevention of infectious diseases, tumors, or allergic diseases.

[27] The immune inducer according to any one of [1] to

[20] for use in the treatment or prevention of tumors.

[28] Use of the immune inducer according to any one of [1] to

[20] for the manufacture of a medicament for the treatment or prevention of infectious diseases, tumors, or allergic diseases. Use of the immune inducer according to any one of [1] to

[20] for the manufacture of a medicament for treating or preventing tumors.

[30] A polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof, wherein the polynucleotide-peptide conjugate comprises a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, a peptide, and a spacer covalently bonded to the polynucleotide or polynucleotide derivative at one end and covalently bonded to the peptide at the other end, wherein the peptide is a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced with amino acids having a reactive functional group for forming a covalent bond with the spacer, and wherein the one or more consecutive amino acids do not contain an anchor residue for MHC binding, the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof.

[31] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to

[30] , wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide are cleavable covalent bonds in a biological environment, wherein the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs, wherein the polynucleotide or polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bond is replaced with a phosphorothioate bond, the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof.

[32] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to

[30] , wherein the amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an analogue thereof having a thiol group, The covalent bond between the spacer and the peptide is a disulfide bond, the MHC-binding peptide is an MHC-I-binding peptide, the MHC-I-binding peptide is an HLA-A-binding peptide or an HLA-B-binding peptide, the amino acid length of the MHC-I-binding peptide is 8 or more and 11 or less, the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs, the base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less, in the polynucleotide derivative in which at least a part of the phosphodiester bond is substituted with a phosphorothioate bond, 90% or more of the phosphodiester bond is substituted with a phosphorothioate bond, the spacer has a structure represented by any of the following formulas, the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof. [Chemical formula]

[0009] The present invention also provides the following [A1] to [A19]. [A1] A polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof, wherein the polynucleotide-peptide conjugate comprises a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, a peptide, and a spacer covalently bonded to the polynucleotide or polynucleotide derivative at one end and covalently bonded to the peptide at the other end, The peptide is a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced with amino acids having a reactive functional group for forming a covalent bond with the spacer, wherein the one or more consecutive amino acids do not include an anchor residue for MHC binding, the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof. [A2] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to [A1], wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide are covalent bonds cleavable in a biological environment. [A3] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to A1 or A2, wherein the amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an analog thereof having a thiol group. [A4] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of A1 to A3, wherein the covalent bond between the spacer and the peptide is a disulfide bond. [A5] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of A1 to A4, wherein the MHC-binding peptide is an MHC-1-binding peptide. [A6] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to A5, wherein the MHC-1-binding peptide is an HLA-A-binding peptide or an HLA-B-binding peptide. [A7] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to A5 or A6, wherein the amino acid length of the MHC-1-binding peptide is 8 or more and 11 or less. [A8] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of A1 to A4, wherein the MHC-binding peptide is an MHC-2-binding peptide. [A9]The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of A1 to A8, wherein the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs. [A10]The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of A1 to A9, wherein the base length of the polynucleotide or polynucleotide derivative is 15 or more and 40 or less. [A11]The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to A10, wherein the base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less. [A12]The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of A1 to A11, wherein the polynucleotide or polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bonds are replaced with phosphorothioate bonds. [A13]The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to A12, wherein in the polynucleotide derivative in which at least a part of the phosphodiester bonds are replaced with phosphorothioate bonds, 50% or more of the phosphodiester bonds are replaced with phosphorothioate bonds. [A14]The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to A13, wherein in the polynucleotide derivative in which at least a part of the phosphodiester bonds are replaced with phosphorothioate bonds, 90% or more of the phosphodiester bonds are replaced with phosphorothioate bonds. [A15]The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of A1 to A14, wherein the spacer contains a repeating unit represented by the following formula. [Chemical formula] In the above formula, X represents an oxygen atom or a sulfur atom (where each X may be the same or different), R is (CH2) p O, (CH2) q NH and (CH2CH2O) m each representing one of them (m, p, and q each independently represent a natural number of 10 or less). n represents a natural number of 10 or less. [A16] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of A1 to A15, wherein the spacer has a structure represented by any of the following formulas.

Chemical formula

Chemical formula

[0010] The present invention also provides the following [A20] and [A21]. [A20] A method for producing an immunomodulator containing a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof as an active ingredient, (1) Preparing a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, (2)Preparing a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced with amino acids having a reactive functional group for forming a covalent bond with the spacer, wherein the one or more consecutive amino acids do not contain an anchor residue, and (3)Linking the polynucleotide or polynucleotide derivative of (1) and the peptide of (2) via a spacer, wherein the spacer covalently binds to the polynucleotide or polynucleotide derivative at one end and covalently binds to the peptide at the other end, A method comprising: [A21] The method according to [A20], wherein the immune inducer is the immune inducer according to any one of [1] to

[20] ,

[26] , and

[27] .

[0011] The present invention also provides the following [A22] and [A23]. [A22] A method for producing a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof, comprising: (1) Preparing a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif; (2) Preparing a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced with amino acids having a reactive functional group for forming a covalent bond with the spacer, wherein the one or more consecutive amino acids do not contain an anchor residue, and (3) Linking the polynucleotide or polynucleotide derivative of (1) and the peptide of (2) via a spacer, wherein the spacer covalently binds to the polynucleotide or polynucleotide derivative at one end and covalently binds to the peptide at the other end, A method comprising: [A23] The method according to [A22], wherein the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof is the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to any one of [A1] to [A19].

[0012] The present invention also provides the following [a1] to [a19]. [a1] An immunostimulant comprising a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the polynucleotide-peptide conjugate comprises a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, a peptide, and a spacer covalently bonded to the polynucleotide or polynucleotide derivative at one end and covalently bonded to the peptide at the other end, wherein the peptide is a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced with amino acids having a reactive functional group for forming a covalent bond with the spacer, and wherein the one or more consecutive amino acids do not include an anchor residue for MHC binding, the immunostimulant. [a2] The immunostimulant according to [a1], wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide are covalent bonds cleavable in a biological environment. [a3] The immunostimulant according to [a1] or [a2], wherein the amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an analog thereof having a thiol group. [a4] The immunostimulant according to any one of [a1] to [a3], wherein the covalent bond between the spacer and the peptide is a disulfide bond. [a5] The immunostimulant according to any one of [a1] to [a4], wherein the MHC-binding peptide is an MHC-1 binding peptide. [a6] The immunostimulant according to [a5], wherein the MHC-1 binding peptide is an HLA-A binding peptide or an HLA-B binding peptide. [a7] The immunostimulant according to [a5] or [a6], wherein the amino acid length of the MHC-1 binding peptide is 8 or more and 11 or less. [a8] The immunostimulant according to any one of [a1] to [a4], wherein the MHC-binding peptide is an MHC-2 binding peptide. [a9]The immunopotentiator according to any one of [a1] to [a8], wherein the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs. [a10]The immunopotentiator according to any one of [a1] to [a9], wherein the base length of the polynucleotide or polynucleotide derivative is 15 or more and 40 or less. [a11]The immunopotentiator according to [a10], wherein the base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less. [a12]The immunopotentiator according to any one of [a1] to [a11], wherein the polynucleotide or polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bonds are substituted with phosphorothioate bonds. [a13]The immunopotentiator according to [a12], wherein in the polynucleotide derivative in which at least a part of the phosphodiester bonds are substituted with phosphorothioate bonds, 50% or more of the phosphodiester bonds are substituted with phosphorothioate bonds. [a14]The immunopotentiator according to [a13], wherein in the polynucleotide derivative in which at least a part of the phosphodiester bonds are substituted with phosphorothioate bonds, 90% or more of the phosphodiester bonds are substituted with phosphorothioate bonds. [a15]The immunopotentiator according to any one of [a1] to [a14], wherein the spacer contains a repeating unit represented by the following formula.

Chemical formula

Chemical formula

Advantages of the Invention

[0013] According to the present invention, a wide range of antigenic peptides can be used for producing an immunopotentiator capable of inducing CTL activity.

Brief Description of the Drawings

[0014]

Figure 1

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Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Mode for Carrying Out the Invention

[0015] The present invention provides an immune inducer containing a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof as an active ingredient (hereinafter, also referred to as the immune inducer of the present invention).

[0016] The polynucleotide-peptide conjugate in the immune inducer of the present invention comprises a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, a peptide, and a spacer covalently bonded to the polynucleotide or polynucleotide derivative at one end and covalently bonded to the peptide at the other end.

[0017] "Single-stranded polynucleotide or polynucleotide derivative containing CpG motif" can be used without particular limitation for polynucleotides or their derivatives having any base sequence and number of bases, as long as they contain one or more (preferably a plurality, for example, 2, 3, 4, 5 or 6) CpG motifs. Specific examples of CpG motifs include AGCGTT, GACGTT, GACGTC, GTCGTT, etc. A plurality of CpG motifs with different sequences may be included. The number of CpG motifs contained in the polynucleotide is not particularly limited, but preferably contains 1 to 6 CpG motifs, and more preferably contains 2 to 4 CpG motifs. The polynucleotide or polynucleotide derivative is preferably a polydeoxyribonucleotide (DNA) or a phosphorothioate-modified DNA derivative containing 2 or more CpG motifs, but may partly contain RNA or RNA derivatives. When RNA or RNA derivatives are included, their content is preferably 20% or less (specifically, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% or less) in terms of the ratio of the number of bases.

[0018] The number of bases contained in the polynucleotide or polynucleotide derivative is preferably 15 to 40 (specifically, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40), and more preferably 20 to 30 (specifically, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30). Specific examples of the base sequences of preferred polynucleotides or polynucleotide derivatives include those shown in Table 1 below. In Table 1, the underlined sequences represent CpG motifs.

[0019]

Table 1-1

[0020]

Table 1-2

[0021] Since polynucleotides are liable to be degraded by nucleases in vivo, polynucleotide derivatives may be used instead of polynucleotides in order to improve their stability in vivo. Polynucleotide derivatives can include any modifications known in the art as modifications for increasing nuclease resistance and enhancing stability in vivo. Examples of polynucleotide derivatives include those in which all or part of the hydroxyl groups at the 2'-position of ribonucleotides are substituted with fluorine or methoxy groups, and those in which all or part of the phosphodiester bonds of polyribonucleotides (RNA) or polydeoxyribonucleotides (DNA) are substituted with phosphorothioate bonds. When part of the phosphodiester bonds of polyribonucleotides or polydeoxyribonucleotides are substituted with phosphorothioate bonds, it is preferable that 50% or more (specifically, 50, 60, 70, 80, or 90% or more) of the phosphodiester bonds are substituted with phosphorothioate bonds, more preferably 90% or more (specifically, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more), and it may even be substantially all substituted with phosphorothioate bonds. All of the phosphodiester bonds may be substituted with phosphorothioate bonds. The positions of the phosphodiester bonds substituted with phosphorothioate bonds are not particularly limited, and a plurality of consecutive phosphodiester bonds may be substituted, or the phosphorothioate bonds may be substituted so as not to be adjacent to each other.

[0022] The position at which the polynucleotide or polynucleotide derivative binds to the spacer is not particularly limited and can be, for example, the 5'-end or the 3'-end. For the covalent bond between the polynucleotide or polynucleotide derivative and the spacer, the functional groups present in the polynucleotide or polynucleotide derivative can be used as they are, or those activated by chemical modification can be used. It is preferable that the polynucleotide or polynucleotide derivative is bound to the spacer on the oxygen atom of the hydroxyl group at its 5'-end or 3'-end.

[0023] The polynucleotide or polynucleotide derivative can be synthesized using known chemical synthesis methods (for example, the triester method, the phosphoramidite method, the H-phosphonate method, etc.). It may also be synthesized using a commercially available nucleic acid synthesizer and reagents used for commercially available DNA / RNA synthesis.

[0024] The "peptide" in the immune inducer of the present invention is a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced with amino acids having a reactive functional group for forming a covalent bond with the spacer, where the one or more consecutive amino acids do not include an anchor residue for MHC binding.

[0025] In the present invention, the "MHC-binding peptide" refers to a peptide that can bind to an MHC molecule (i.e., the major histocompatibility complex) without additional processing such as trimming and can be presented as an antigen to T cells. MHC molecules include MHC-1 molecules (also referred to as MHC class I molecules) and MHC-2 molecules (also referred to as MHC class II molecules). In humans, MHC-1 molecules are called HLA molecules and there are many alleles. The MHC-binding peptide is preferably either a peptide presented by MHC-1 molecules (i.e., an MHC-1 binding peptide) or a peptide presented by MHC-2 molecules (i.e., an MHC-2 binding peptide), more preferably an MHC-1 binding peptide, and even more preferably a peptide presented by HLA-A molecules (i.e., an HLA-A binding peptide) or a peptide presented by HLA-B molecules (i.e., an HLA-B binding peptide).

[0026] The MHC-binding peptide can be derived from proteins that cause allergies such as food allergies, pathogens such as bacteria and viruses, and proteins originating from tumor cells and the like. Examples of MHC-binding peptides include, but are not limited to, peptides listed in the public database SYFPEITHI (see http: / / www.syfpeithi.de / 0-Home.htm; Immunogenetics (1999) 50:213-219) and peptides listed in the table of Immunogenetics (1995) 41:178-228. Specific examples of MHC-1 binding peptides include OVA peptide 1 (a peptide consisting of the amino acid sequence at positions 258 to 265 of ovalbumin (OVA; GenBank accession number: CAA23716.1), SIINFEKL (SEQ ID NO: 41)), TRP2-9 (a peptide consisting of the amino acid sequence at positions 180 to 188 of mTRP2 (GenBank accession number: CAA44951.1)), hGP100-9 (a peptide consisting of the amino acid sequence at positions 25 to 33 of hGP100 (GenBank accession number: AAC60634.1)), and the like. Specific examples of MHC-2-binding peptides include OVA peptide 2 (a peptide consisting of the amino acid sequence at positions 324 to 340 of OVA, ISQAVHAAHAEINEAGR (SEQ ID NO: 42)).

[0027] In MHC molecules, there is a site called a pocket that directly interacts with the MHC-binding peptide within the peptide-binding groove. In MHC-binding peptides, the residues that interact with a specific pocket are called anchor residues or anchor amino acids, and their positions can vary depending on the type of MHC molecule to which they bind. The positions of the anchor residues and the common motifs for each type of MHC molecule can be confirmed, for example, by the public database MHC Motif Viewer (see http: / / www.cbs.dtu.dk / biotools / MHCMotifViewer / Home.html; Immunogenetics (2008) 60:759-765). For example, in HLA-A and HLA-B, which are human MHC-1, the second and ninth amino acids from the N-terminal side are known to be anchor residues.

[0028] In addition to the peptides that can exist naturally (wild-type peptides) as described above, MHC-binding peptides also include peptides that do not exist naturally, for example, peptides containing non-natural amino acids. Examples of such peptides include modified peptides (called heteroclitic peptides) in which the anchor site has been substituted with other amino acids (including non-natural amino acids) to enhance the binding affinity to MHC molecules (see Front. Immunol. 6:377 (2015) and J Immunol. 174(8):4812-4820 (2005)).

[0029] The amino acid length of the MHC-binding peptide is not particularly limited and may vary depending on the type of MHC molecule, but can be, for example, 5 or more and 30 or less. The length of the peptide that binds to MHC-1 molecules is somewhat fixed and is usually 8 to 11 amino acids in length. Therefore, in the present invention, when the MHC-binding peptide is an MHC-1 binding peptide, its amino acid length is preferably 8 or more and 11 or less, more preferably 8, 9 or 10, and even more preferably 9 or 10.

[0030] The inventors of the present invention found that adding an amino acid such as cysteine to the N-terminus of an MHC-binding peptide for conjugation may prevent it from binding to MHC molecules (Example 3). It is known that endoplasmic reticulum aminopeptidase (ERAP) is involved in the presentation of antigenic peptides by MHC-1 molecules, and antigenic peptide precursors usually undergo trimming from the N-terminus by ERAP to a length suitable for binding to MHC-1. However, for peptides generated from polynucleotide-peptide conjugates, such trimming may not be appropriately carried out, and it is possible that they cannot form the structure of the original peptide having the ability to bind to the peptide-binding groove of MHC molecules. In contrast, a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced with an amino acid having a reactive functional group for forming a covalent bond with the spacer while leaving the anchor residue can bind to MHC-1 molecules. In addition, a polynucleotide-peptide conjugate prepared using the peptide has sufficient CTL-inducing ability.

[0031] The number of amino acids at the N-terminus of the MHC-binding peptide substituted with an amino acid having a reactive functional group is not particularly limited as long as it does not include an anchor residue for MHC binding, and can be appropriately set according to the type of MHC molecule. For example, in the case of MHC-I molecules, the number of substituted amino acids can be 4 or less (4, 3, 2, or 1), and 1 is preferred. In one embodiment, when the MHC-binding peptide is an HLA-A-binding peptide or an HLA-B-binding peptide, since the anchor residue is present at the second position from the N-terminus, the number of substituted amino acids can be 1.

[0032] The "amino acid having a reactive functional group for forming a covalent bond with the spacer" is an amino acid having a reactive functional group capable of forming a covalent bond such as an ester bond, an amide bond, or a phosphate ester bond with the spacer, and may be either a natural amino acid or a non-natural amino acid. The covalent bond with the spacer is preferably a covalent bond that can be cleaved in the in vivo environment. Examples of such covalent bonds include bonds that are cleaved in the reducing environment inside cells, such as disulfide bonds. Also, ester bonds, amide bonds (e.g., amide bonds with cathepsin-sensitive peptides, etc.), and phosphodiester bonds that are specifically cleaved by intracellular enzymes such as esterases, peptidases (e.g., cathepsin, etc.), and nucleases are also examples. As a more preferred embodiment, the covalent bond is a disulfide bond, and the reactive functional group can be a thiol group. In this case, the "amino acid having a reactive functional group for forming a covalent bond with the spacer" can be cysteine or an analog thereof having a thiol group. A cysteine analog having a thiol group is an amino acid having a thiol in its side chain, and the structure of the side chain is not particularly limited. Examples of cysteine analogs having a thiol group include homocysteine (CAS No: 6027-13-0), penicillamine (β,β-dimethylcysteine; CAS No: 1113-41-3), β-methylcysteine (CAS No: 29768-80-7), and 4-mercapto-norvaline (CAS No: 2351397-00-5).

[0033] The peptide in the immunopotentiator of the present invention can be prepared using any known method such as peptide synthesis.

[0034] The "spacer" in the immunopotentiator of the present invention may be a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif and a structure capable of covalently binding to a peptide, and examples thereof include an alkylene group and polyethylene glycol (PEG). The spacer may contain a repeating unit including a phosphodiester structure or phosphorothioate structure represented by the following formula.

[0035]

Chemical formula

[0036] In the above formula, X represents an oxygen atom or a sulfur atom (where each X may be the same or different), and R represents (CH2) p O, (CH2) q NH, (CH2CH2O) m any of them (where m, p, and q each independently represent a natural number of 10 or less), and n represents a natural number of 10 or less.

[0037] Since these repeating units are not hydrolyzed by nucleases, even if X is an oxygen atom, the stability in vivo does not significantly decrease. For example, when R is (CH2)3O, the size of the repeating unit becomes approximately equal to the size of ribonucleotides or deoxyribonucleotides, so a reduction in production cost can be expected by substituting a part of the polynucleotide or polynucleotide derivative with this spacer. Specific examples of the spacer include the following.

[0038]

Chemical formula

[0039]

Chemical formula

[0040]

Chem.

[0041]

Chem.

[0042]

Chem.

[0043]

Chem.

[0044]

Chem.

[0045]

Chem.

[0046] More preferred examples of the spacer include those having any of the following structures.

[0047]

Chem.

[0048] In another aspect, more preferred examples of the spacer include those having any of the following structures.

[0049]

Chem.

[0050] Examples of combinations of reactive functional groups used for forming a bond between a spacer and a polynucleotide or polynucleotide derivative and for forming a bond between a spacer and a peptide include, in addition to combinations of reactive functional groups that form an ester bond, an amide bond, a phosphate ester bond, etc., for example, combinations of reactive functional groups used for immobilizing a biomolecule on a biochip surface, and more specifically, those shown below.

[0051] (a) Alkyne and azide compound Alkyne and azide compounds (azides) form a 1,2,3-triazole ring by an addition cyclization reaction (Huisgen reaction) as shown below. Both are stable functional groups that can be introduced into many organic compounds including biomolecules, react rapidly and almost quantitatively even in a solvent containing water, with almost no side reactions and no generation of excess waste, and thus are widely used in the field of biochemistry as a central reaction of so-called "click chemistry". An alkyne derivative and an azide group can be introduced into an antigenic peptide or polynucleotide or polynucleotide derivative using any known method. As alkyne derivatives, those having reactive functional groups such as propargyl alcohol and propargylamine are readily available, and these can be directly reacted with reactive functional groups such as a carboxyl group or a hydroxyl group, or reacted with carbonyldiimidazole etc., and the alkyne derivative can be introduced via an amide bond, ester bond, urethane bond, etc. formed. Regarding the azide group, it can also be introduced into an antigenic peptide or polynucleotide or polynucleotide derivative using any known method. Note that the Huisgen reaction is carried out in the presence of a copper catalyst, but for an antigenic peptide and a polynucleotide derivative in which a phosphodiester bond is substituted with a sulfur-containing functional group such as a phosphorothioate bond, since there is a sulfur atom that coordinates to copper ions, the catalytic activity of copper may decrease. It is preferable to add an excessive amount of copper to improve the reaction rate.

[0052] [Chemical formula]

[0053] (b) Maleimide or vinyl sulfone and thiol group Maleimides or vinyl sulfones having a double bond adjacent to an electron-withdrawing carbonyl group or sulfone group form stable thioether derivatives by an addition reaction (Michael addition reaction) with a thiol group as shown below at a pH near neutrality. Since maleimide and vinyl sulfone derivatives having appropriate spacers are commercially available, it is easy to introduce these functional groups into a peptide, polynucleotide or polynucleotide derivative having antigenicity. When introducing a thiol group into a peptide having antigenicity, in the case of a peptide having antigenicity containing cysteine, the thiol group of the cysteine residue side chain can be utilized. However, since cysteine is an amino acid with a low abundance ratio, a peptide having cysteine introduced at the N-terminal side of the peptide having antigenicity is used. As the polynucleotide or polynucleotide derivative containing a thiol group, a thiolated polynucleotide obtained by converting the hydroxyl group at the 5'-end thereof into a thiol group is used.

[0054] [Chemical formula]

[0055] (c) Thiol group of peptide and thiol group of thiolated polynucleotide As described above, the thiol group introduced at the N-terminal of the peptide is reacted with the thiol group of the thiolated polynucleotide to form a disulfide group. Since the disulfide bond is cleaved in the presence of a reducing agent, it is inferior in terms of stability compared to the above two, but has the advantage of being cleaved in a reducing environment in vivo. The introduction of a thiol group into a polynucleotide or polynucleotide derivative can be carried out using any known method. Specific examples include the reaction of an aminoated polynucleotide or polynucleotide derivative with an N-succinimidyl ester of ω-(2-pyridyldithio) fatty acid as shown in the following formula.

[0056] [Chem.]

[0057] One or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide is a covalent bond cleavable in a biological environment, and it is preferable that at least the covalent bond between the spacer and the peptide is a covalent bond cleavable in a biological environment. Therefore, among the above (a) to (c), a disulfide bond formed by the combination of the thiol group of a peptide easily cleavable in vivo and the thiol group of a thiolated polynucleotide is preferable. In this case, the disulfide bond is a covalent bond between the spacer and the peptide.

[0058] Specific examples of the polynucleotide-peptide conjugate in the immunomodulator of the present invention include CpG30(S)a-mTRP2pep9 (Compound 1), CpG20(S)a-mTRP2pep9 (Compound 2), and CpG30(S)a-hGP100pep9 (Compound 3) described in Example 1 below, and the following compounds. In the following formula, the part represented by the base sequence indicates a DNA derivative in which the phosphodiester bond is replaced by a phosphorothioate bond.

[0059] [Chem.]

[0060] Specific examples of the polynucleotide-peptide conjugate in the immunomodulator of the present invention also include ISS1018-mTRP2pep9, ODN2006-mTRP2pep9, and ODN1826-mTRP2pep9 described in Example 5 below, CpG30(S)a2-OVApep8 and CpG30(S)a2-mTRP2pep9 described in Example 6, and CpG30(S)a2-OVA2-15 described in Example 9.

[0061] In one aspect, one polynucleotide or polynucleotide derivative contained in the immunogenic agent of the present invention may be bound to two or more peptides. That is, the immunogenic agent of the present invention may contain one polynucleotide or polynucleotide derivative and two or more peptides. The polynucleotide or polynucleotide derivative and two or more peptides may be bound via a spacer. The polynucleotide or polynucleotide derivative can be bound to two or more peptides via, for example, separate spacers respectively. The polynucleotide or polynucleotide derivative may be bound to two or more peptides via a branched spacer. One polynucleotide or polynucleotide derivative may be bound to three or more peptides by a combination of these binding modes. The position where the polynucleotide or polynucleotide derivative moiety binds to the spacer is not particularly limited and can be selected, for example, from the 5'-end and the 3'-end. The two or more peptides may be the same peptide or different peptides, but it is preferable that they are all the same. The number of peptides bound to one polynucleotide or polynucleotide derivative is not particularly limited and can be, for example, two, three, four, five, or more.

[0062] In one aspect, the immunogenic agent of the present invention may form a double strand with a polynucleotide or polynucleotide derivative having a base sequence complementary to the base sequence contained in the polynucleotide or polynucleotide derivative moiety (hereinafter also referred to as a complementary strand polynucleotide or polynucleotide derivative) in the polynucleotide or polynucleotide derivative moiety. The complementary strand polynucleotide or polynucleotide derivative can be synthesized in the same manner as the polynucleotide or polynucleotide derivative portion in the immunomodulator of the present invention. The complementary strand polynucleotide or polynucleotide derivative may be modified for improving properties such as stability in vivo, toxicity, and pharmacokinetics. Such modifications include, for example, lipid modification (see WO2017 / 057540). Compounds having lipid modification at one or both of the 5' or 3' ends can be synthesized by the method described in Example 8. The double-strand formation between the polynucleotide or polynucleotide derivative portion of the immunomodulator of the present invention and the complementary strand polynucleotide or polynucleotide derivative can be carried out according to a general annealing method. For example, a polynucleotide-peptide conjugate and a complementary strand polynucleotide or polynucleotide derivative are mixed, heated to a single-stranded state, and then allowed to cool naturally to room temperature to form a double strand.

[0063] Examples of pharmaceutically acceptable salts of the polynucleotide-peptide conjugate used as an active ingredient of the immunomodulator of the present invention include salts of alkali metals (such as potassium, sodium, and lithium), salts of alkaline earth metals (such as calcium and magnesium), ammonium salts (including tetramethylammonium salts and tetrabutylammonium salts), salts of organic amines (such as triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)methylamine, lysine, arginine, and N-methyl-D-glucamine), and acid addition salts (inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, and nitrate; and organic acid salts such as acetate, trifluoroacetate, lactate, tartrate, oxalate, fumarate, maleate, benzoate, citrate, methanesulfonate (mesylate), ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate, and gluconate).

[0064] The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof used as an active ingredient of the immunopotentiator of the present invention may exist as a solvate (including hydrate). The solvate is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include hydrates, ethanolates, and the like.

[0065] The immunomodulator of the present invention may further contain a substance having immunostimulatory activity as an adjuvant. The adjuvant is, but not limited to, a substance that activates innate immunity. The adjuvant is preferably an agonist of an innate immune receptor. Examples of innate immune receptor agonists include TLR agonists (e.g., TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR7 agonist, TLR8 agonist, TLR9 agonist), RLR (retinoic acid-inducible gene I (RIG-1)-like receptors) agonists, STING (stimulator of Interferon genes) agonists, NLR (nucleotide-binding oligomerization domain (NOD)-like receptors) agonists, CLR (C-type lectin receptors) agonists, etc. Examples of TLR agonists include lipopeptides, Poly IC RNA, imiquimod, resiquimod, monophosphoryl lipid A (MPL), CpG-ODN, etc. Examples of RLR agonists include pppRNA, Poly IC RNA, etc., examples of STING agonists include cGAMP, c-di-AMP, c-di-GMP, etc., examples of NLR agonists include iE-DAP, FK565, MDP, murabutide, etc., and examples of CLR agonists include beta-glucan, trehalose 6,6'-dimycolate, etc. The adjuvant is preferably a TLR agonist, more preferably a TLR4 agonist, TLR7 agonist or TLR9 agonist, and even more preferably imiquimod, resiquimod, MPL or CpG-ODN. In one embodiment, the adjuvant is imiquimod, MPL or CpG-ODN. The adjuvant can be appropriately selected according to, for example, peptides introduced into polynucleotide-peptide conjugates, etc., and may be, for example, CpG DNA, or a polynucleotide / β-1,3-glucan complex described in International Publication No. 2015 / 118789.

[0066] The immunomodulator of the present invention (1)Preparing a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif; (2)Preparing a peptide in which one or more consecutive amino acids at the N-terminus of an MHC-binding peptide are replaced with amino acids having a reactive functional group for forming a covalent bond with the spacer, wherein the one or more consecutive amino acids do not contain an anchor residue, and (3)Linking the polynucleotide or polynucleotide derivative of (1) and the peptide of (2) via a spacer, wherein the spacer covalently binds to the polynucleotide or polynucleotide derivative at one end and covalently binds to the peptide at the other end; It can be produced by a method comprising the above. Therefore, in one aspect, the present invention provides a method for producing an immunogenic inducer of the present invention, which includes the above steps (1) to (3). Each term in this aspect is interpreted based on the description of the terms described herein. By this method, an immunogenic inducer capable of inducing CTL activity can be produced using a wide range of antigenic peptides.

[0067] The present invention also provides a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof. Each term in this aspect is interpreted based on the description of the terms described herein. The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof can be used as an active ingredient in the immunogenic inducer of the present invention. The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof can also be produced by a method including the above steps (1) to (3) in the method for producing the immunogenic inducer of the present invention. Therefore, in one aspect, the present invention provides a method for producing a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof, which includes the above steps (1) to (3). Each term in this aspect is interpreted based on the description of the terms described herein. By this method, a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof that can be an active ingredient of an immunogenic inducer capable of inducing CTL activity can be produced using a wide range of antigenic peptides.

[0068] The present invention also provides a pharmaceutical composition comprising the immunopotentiator of the present invention (hereinafter also referred to as the pharmaceutical composition of the present invention). In the production of the pharmaceutical composition of the present invention, in addition to the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof as an active ingredient, any known ingredients (any carrier, excipient and additive acceptable for pharmaceutical use) and formulation methods can be used. Examples of substances for formulation include, but are not limited to: amino acids such as glycine, alanine, glutamine, asparagine, arginine or lysine; antioxidants such as ascorbic acid, sodium sulfate or sodium bisulfite; buffers such as phosphoric acid, citric acid, boric acid buffer, sodium bicarbonate, Tris-hydrochloride (Tris-HCl) solution; fillers such as mannitol and glycine; chelating agents such as ethylenediaminetetraacetic acid (EDTA); complexing agents such as caffeine, polyvinylpyrrolidine, β-cyclodextrin and hydroxypropyl-β-cyclodextrin; bulking agents such as glucose, mannose or dextrin; other carbohydrates such as monosaccharides and disaccharides; coloring agents; flavoring agents; diluents; hydrophilic polymers such as emulsifiers and polyvinylpyrrolidine; low molecular weight polypeptides; salt-forming counterions; preservatives such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine, sorbic acid or hydrogen peroxide; solvents such as glycerin, propylene glycol or polyethylene glycol; sugar alcohols such as mannitol or sorbitol; suspending agents; sorbitan esters; polysorbates such as polysorbate 20 and polysorbate 80; surfactants such as triton, tromethamine, lecithin or cholesterol; stabilizing enhancers such as sucrose and sorbitol; elasticity enhancers such as sodium chloride, potassium chloride, mannitol and sorbitol; transport agents; excipients; and / or pharmaceutical adjuvants. Those skilled in the art can appropriately determine the composition of a suitable pharmaceutical composition according to the applicable disease, applicable administration route, etc.

[0069] The pharmaceutical composition of the present invention is provided in dosage forms suitable for oral or parenteral administration. For example, injections, suppositories, etc. are used, and injections can include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. Such injections can be prepared according to known methods. As a method for preparing an injection, for example, the above immunomodulator of the present invention can be prepared by dissolving or suspending it in a sterile aqueous solvent usually used for injections. As the aqueous solvent for injection, for example, distilled water, physiological saline, phosphate buffer, carbonate buffer, Tris buffer, acetate buffer and other buffers can be used. The pH of such an aqueous solvent is preferably 5 to 10, more preferably 6 to 8. The prepared injection solution is preferably filled into an appropriate ampoule. The injection can also be a freeze-dried preparation. In addition to injections, dosage forms for transdermal or mucosal absorption (liquid sprays, ointments, gels, lotions, patches), dosage forms for subcutaneous local sustained release (suspensions containing nanogels and biodegradable micro / nanocapsules, temperature-responsive gels), preparations including transdermal devices for skin permeation (iontophoresis, microneedles), powders, tablets, capsules, syrups, inhalants such as aerosols and dry powders, etc. can be adopted.

[0070] The pharmaceutical composition of the present invention can be administered to humans or warm-blooded animals (such as mice, rats, rabbits, sheep, pigs, cows, horses, chickens, cats, dogs, monkeys, etc.) by either oral or parenteral routes. Parenteral administration routes include subcutaneous, intradermal and intramuscular injections, intraperitoneal administration, drip infusion, intravenous administration, administration to the oral mucosa, spraying onto the nasal mucosa or pharynx, etc.

[0071] The dosage of the polynucleotide-peptide conjugate, which is the active ingredient of the pharmaceutical composition of the present invention, varies depending on factors such as activity, the disease to be treated, the type of animal to be administered, body weight, sex, age, type of disease, and administration method. Taking an adult weighing 60 kg as an example, in the case of oral administration, the daily dosage is usually about 0.1 to about 100 mg, preferably about 1.0 to about 50 mg, more preferably about 1.0 to about 20 mg. In the case of parenteral administration, the daily dosage is usually about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, more preferably about 0.1 to about 10 mg. When administering to other animals, the dosage obtained by multiplying the above dosage converted to the dosage per unit body weight by the body weight of the animal to be administered is used.

[0072] Also, the frequency of administration of the pharmaceutical composition of the present invention can be easily determined by those skilled in the art in consideration of factors such as the type of animal to be administered, body weight, sex, age, type of disease, and administration method. For example, when the composition of the present invention is administered as a vaccine, similar to ordinary vaccine preparations, it can usually be administered 1 to several times a day, either once a day or several times at intervals of 1 to several weeks. Administration is preferably carried out while observing the progress. For example, booster immunization can be carried out at intervals of at least about 1 week. By performing booster immunization, a booster effect is expected to be obtained, and effects such as higher protection against infection can be achieved.

[0073] By administering the pharmaceutical composition of the present invention to patients with pathogen infections or cancer, or to subjects who are at risk of developing cancer or pathogen infections, cytotoxic T cells (CTLs) and helper T cells in the subjects receiving the administration are antigen-specifically activated, and by inducing a defensive immune response in warm-blooded animals (preferably humans), the infections and cancer can be prevented and treated. Further, by administering the pharmaceutical composition of the present invention to patients with allergic diseases, it becomes an antigen-specific immunotherapy that suppresses the excessive immune response against the allergenic antigen that causes the disease. That is, the pharmaceutical composition of the present invention is useful as a vaccine for the prevention or treatment of diseases such as the above-mentioned infections, cancer, and allergic diseases. In the present invention, the terms "tumor" and "cancer" are used interchangeably. Further, in the present invention, tumors, malignant tumors, cancers, malignant neoplasms, carcinomas, sarcomas, etc. may be collectively referred to as "tumor" or "cancer". Further, the terms "tumor" and "cancer" include pathological conditions that are sometimes classified into the precancerous stage, such as myelodysplastic syndrome.

[0074] The type of tumor to be treated or prevented is not particularly limited as long as it is a tumor that has been confirmed to be sensitive to the pharmaceutical composition of the present invention, but breast cancer, colon cancer, prostate cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, etc.), gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine body cancer, kidney cancer, hepatocellular carcinoma, thyroid cancer, esophageal cancer, osteosarcoma, skin cancer (including melanoma, etc.), glioblastoma, neuroblastoma, ovarian cancer, head and neck cancer, testicular tumor, colorectal cancer, blood cancer (including leukemia, malignant lymphoma, multiple myeloma, etc.), retinoblastoma, pancreatic cancer, etc. can be mentioned.

[0075] The pharmaceutical composition of the present invention may be used in combination with other antitumor agents. For example, antitumor antibiotics, antitumor plant components, BRM (Biological Response Modifier), hormones, vitamins, antitumor antibodies, molecular target drugs, alkylating agents, antimetabolites, and other antitumor agents can be mentioned.

[0076] More specifically, examples of antitumor antibiotics include mitomycin C, bleomycin, peplomycin, daunorubicin, aclarubicin, doxorubicin, idarubicin, pirarubicin, THP-adriamycin, 4'-epi-doxorubicin or epirubicin, chromomycin A3, actinomycin D, and the like.

[0077] Examples of antitumor plant components and their derivatives include vinca alkaloids such as vindesine, vincristine or vinblastine, taxanes such as paclitaxel, docetaxel, cabazitaxel, or epipodophyllotoxins such as etoposide or teniposide.

[0078] Examples of BRM include tumor necrosis factor, indomethacin, and the like.

[0079] Examples of hormones include hydrocortisone, dexamethasone, methylprednisolone, prednisolone, plastolone, betamethasone, triamcinolone, oxymetholone, nandrolone, methenolone, phosphoestrol, ethinyl estradiol, chlormadinone, mepetiostane, medroxyprogesterone, and the like.

[0080] Examples of vitamins include vitamin C, vitamin A, and the like.

[0081] Examples of antitumor antibodies and molecular target drugs include trastuzumab, rituximab, cetuximab, panitumumab, nimotuzumab, denosumab, bevacizumab, infliximab, ipilimumab, nivolumab, pembrolizumab, avelumab, pidilizumab, atezolizumab, ramucirumab, imatinib mesylate, dasatinib, sunitinib, lapatinib, dabrafenib, trametinib, cobimetinib, pazopanib, palbociclib, panobinostat, sorafenib, crizotinib, bemrafenib, xalkori, bortezomib, carfilzomib, ixazomib, midostaurin, gilteritinib, and the like.

[0082] Examples of alkylating agents include alkylating agents such as nitrogen mustard, nitrogen mustard N-oxide, bendamustine or chlorambucil, aziridine-based alkylating agents such as carboquone or thiotepa, epoxide-based alkylating agents such as dibromomannitol or dibromodulcitol, nitrosourea-based alkylating agents such as carmustine, lomustine, semustine, nimustine hydrochloride, streptozocin, chloroozotocin or ranimustine, busulfan, improsulfan tosylate, temozolomide or dacarbazine, etc.

[0083] Examples of antimetabolites include purine antimetabolites such as 6-mercaptopurine, 6-thioguanine or thioinosine, pyrimidine antimetabolites such as fluorouracil, tegafur, tegafur-uracil, carmofur, doxifluridine, broxuridine, cytarabine or enocitabine, folic acid antimetabolites such as methotrexate or trimethoprim, etc.

[0084] Examples of other antitumor agents include cisplatin, carboplatin, oxaliplatin, tamoxifen, letrozole, anastrozole, exemestane, toremifene citrate, fulvestrant, bicalutamide, flutamide, mitotane, leuprorelin, goserelin acetate, camptothecin, ifosfamide, cyclophosphamide, melphalan, L-asparaginase, asecratone, sizofiran, picibanil, procarbazine, pipobroman, neocarzinostatin, hydroxyurea, ubenimex, thalidomide, lenalidomide, pomalidomide, eribulin, tretinoin or krestin, etc.

[0085] Examples of the types of infectious diseases to be treated or prevented include infectious diseases caused by pathogens such as viruses, fungi, and bacteria. Examples of viruses include influenza virus, hepatitis virus, human immunodeficiency virus (HIV), respiratory syncytial virus (RS virus), rubella virus, measles virus, mumps virus, herpes virus, poliovirus, rotavirus, Japanese encephalitis virus, varicella virus, adenovirus, rabies virus, yellow fever virus, and the like. Examples of bacteria include Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus pneumoniae, Helicobacter pylori, Bacillus anthracis, Salmonella typhi, Neisseria meningitidis, Shigella dysenteriae, Vibrio cholerae, and the like. Examples of fungi include Candida fungi, Histoplasma fungi, Cryptococcus fungi, Aspergillus fungi, and the like. The pharmaceutical composition of the present invention may be used in combination with existing therapeutic agents for these infectious diseases.

[0086] Examples of the types of allergic diseases to be treated or prevented include bronchial asthma, allergic rhinitis, atopic dermatitis, urticaria, food allergy, animal allergy, anaphylaxis, and the like.

[0087] When the pharmaceutical composition of the present invention is administered in combination with an adjuvant or other medicaments, it means that the subject takes both medicaments into its body over a certain period. A formulation in which both medicaments are contained in a single formulation may be administered, or each may be formulated separately and administered separately. When formulated separately, the timing of administration is not particularly limited, and they may be administered simultaneously, at different times with a time interval, or on different days. When administered at different times or on different days, the order of administration is not particularly limited. Usually, since each formulation is administered according to its respective administration method, the number of administrations may be the same or different. Also, when each is formulated separately, the administration method (route of administration) of each formulation may be the same or may be administered by different administration methods (routes of administration). Further, it is not necessary for both medicaments to be present in the body at the same time, and it is sufficient if they are taken into the body within a certain period (for example, one month, preferably one week, more preferably several days, and even more preferably one day), and the other active ingredient may disappear from the body at any time of administration.

[0088] In another aspect, the present invention provides a method for treating or preventing a disease, which comprises administering a therapeutically or prophylactically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof. Examples of the disease include, for example, infectious diseases caused by pathogens, tumors, and allergic diseases, and preferably tumors.

[0089] The "therapeutically or prophylactically effective amount" in the present invention means an amount that exhibits a therapeutic or prophylactic effect for a specific disease or symptom, administration form, and administration route, and is appropriately determined according to the species of the subject, the type of the disease or symptom, the symptom, sex, age, pre-existing disease, and other factors.

[0090] This application also provides the following inventions. [B1] An immunomodulator containing a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof as an active ingredient, The polynucleotide-peptide conjugate consists of a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, a peptide, and a spacer that is covalently bound to the polynucleotide or polynucleotide derivative at one end and covalently bound to the peptide at the other end. The peptide is a peptide in which one or more consecutive amino acids at the N-terminus and / or C-terminus of the MHC-2 binding peptide have been deleted, and an amino acid having a reactive functional group for forming a covalent bond with the spacer has been added, where the one or more consecutive amino acids do not include an anchor residue for MHC-2 binding, the immune inducer. [B2] The immune inducer according to [B1], wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide are covalent bonds that are cleavable in a biological environment. [B3] The immune inducer according to [B1] or [B2], wherein the amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an analog thereof having a thiol group. [B4] The immune inducer according to any one of [B1] to [B3], wherein the covalent bond between the spacer and the peptide is a disulfide bond. [B5] The immune inducer according to any one of [B1] to [B4], wherein the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs. [B6] The immune inducer according to any one of [B1] to [B5], wherein the base length of the polynucleotide or polynucleotide derivative is 15 or more and 40 or less. [B7] The immune inducer according to [B6], wherein the base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less. [B8] The immune inducer according to any one of [B1] to [B7], wherein the polynucleotide or polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bond is replaced by a phosphorothioate bond. [B9]In the polynucleotide derivative in which at least a part of the phosphodiester bonds are replaced with phosphorothioate bonds, an immunopotentiator according to [B8], wherein 50% or more of the phosphodiester bonds are replaced with phosphorothioate bonds. [B10]In the polynucleotide derivative in which at least a part of the phosphodiester bonds are replaced with phosphorothioate bonds, an immunopotentiator according to [B9], wherein 90% or more of the phosphodiester bonds are replaced with phosphorothioate bonds. [B11]An immunopotentiator according to any one of [B1] to [B10], wherein the spacer contains a repeating unit represented by the following formula. [Chemical formula] In the above formula, X represents an oxygen atom or a sulfur atom (where each X may be the same or different), R is (CH2) p O, (CH2) q NH and (CH2CH2O) m represents any one of them (m, p and q each independently represent a natural number of 10 or less). n represents a natural number of 10 or less. [B12]An immunopotentiator according to any one of [B1] to [B11], wherein the spacer has a structure represented by any of the following formulas. [Chemical formula] [B13] An immunopotentiator according to any one of [B1] to [B10], wherein the spacer has a structure represented by any of the following formulas. [Chemical formula] [B14]An immunopotentiator according to any one of [B1] to [B13], further comprising a substance having immunostimulatory activity as an adjuvant. [B15]A pharmaceutical composition comprising an immunopotentiator according to any one of [B1] to [B14]. [B16] The pharmaceutical composition according to [B15], which is for the treatment or prevention of an infectious disease, a tumor, or an allergic disease. [B17] A method for treating or preventing an infectious disease, a tumor, or an allergic disease, which comprises administering to a patient an immune inducer described in any one of [B1] to [B14]. [B18] An immune inducer described in any one of [B1] to [B14] for use in the treatment or prevention of an infectious disease, a tumor, or an allergic disease. [B19] Use of an immune inducer described in any one of [B1] to [B14] for the manufacture of a pharmaceutical composition for the treatment or prevention of an infectious disease, a tumor, or an allergic disease.

[0091] In the following description, the inventions of [B1] to [B19] above are referred to as Invention B of the present invention. Each term in Invention B of the present invention is interpreted based on the description of the terms described in this specification, as long as it does not conflict with the invention using the MHC-2 binding peptide.

[0092] In Invention B of the present invention, the peptide contained in the polynucleotide-peptide conjugate has one or more consecutive amino acids deleted from the N-terminus and / or C-terminus of the MHC-2 binding peptide, and an amino acid having a reactive functional group for forming a covalent bond with the spacer is added. The number of amino acids to be deleted is not particularly limited as long as it does not include the anchor residue for MHC-2 binding, and can be appropriately set according to the length of the original MHC-2 binding peptide and the position of the anchor residue. The amino acid having a reactive functional group for forming a covalent bond with the spacer can be added to the N-terminus or C-terminus.

[0093] In Invention B of the present invention, the amino acid length of the peptide contained in the polynucleotide-peptide conjugate is not particularly limited, and can be, for example, 8 or more and 30 or less.

[0094] Specific examples of the immune inducer in Invention B of the present invention include, for example, CpG30(S)a2-OVA2-15 described in Example 9 below.

[0095] According to Invention B of the present invention, since a polynucleotide-peptide conjugate can be prepared using a shorter peptide, a pharmaceutical composition having more excellent physical properties and / or economic efficiency can be provided.

Examples

[0096] Next, examples conducted to confirm the effects of the present invention will be described. In this example, "CpG DNA(S)" represents a DNA derivative (an example of a polynucleotide derivative) having a base sequence containing a CpG motif and in which a phosphodiester bond is replaced with a phosphorothioate bond. In the examples, the polynucleotide derivative is represented by a single-letter base sequence listing with the left side being the 5'-terminal side (the right side being the 3'-terminal side), and the peptide is represented by a single-letter notation with the left side being the N-terminal side (the right side being the C-terminal side). In the polynucleotide derivative represented by the single-letter base sequence listing, all phosphodiester bonds are replaced with phosphorothioate bonds, and the terminal structure means up to the oxygen atom of the 5' or 3'-hydroxyl group of the terminal nucleoside when bound to a spacer, or the 5' or 3'-hydroxyl group (including the hydrogen atom) of the terminal nucleoside when not bound to a spacer. Also, the CpG DNA(S)-peptide conjugate in this example is prepared as a salt to which triethylamine and acetic acid are added.

[0097] Example 1: Preparation of CpG DNA(S)-peptide conjugate (1) Synthesis of CpG DNA(S) derivative The synthesis of CpG DNA(S) was carried out using the phosphoramidite method (for example, Nucleic Acids Research, 12, 4539 (1984)). The synthesis of CpG DNA(S) having an amino group modification was carried out using ssH Amino Linker (Bioorg. Med. Chem., 16, 941-949 (2008)). For these syntheses, a contract synthesis service (Gene Design Co., Ltd.) was utilized. The nucleotide sequences of the synthesized CpG DNAs (S) are shown below. CpG30a: 5'-GAGCGTTCTCATCGACTCTCGAGCGTTCTC-3' (K3-30(a) in Table 1; SEQ ID NO: 6) CpG20a: 5'-ATCGACTCTCGAGCGTTCTC-3' (K3 in Table 1; SEQ ID NO: 1)

[0098] The obtained amino group-modified CpG DNAs (S) have a structure represented by the following formula at the 5'-end. Hereinafter, among the CpG DNA (S) derivatives having a structure represented by the following formula at the 5'-end, those having the sequence of SEQ ID NO: 6 are referred to as "CpG30(S)a", and those having the sequence of SEQ ID NO: 1 are referred to as "CpG20(S)a".

[0099]

Chemical formula

[0100] Furthermore, the amino group-modified CpG DNA (S) and succinimidyl 6-[3'-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP) were mixed at a molar ratio of 1:30 in a phosphate buffer (pH 8.0), allowed to stand at 40 °C for 3 hours, and then the SPDP-modified CpG DNA (S) was purified using a NAP-5 column. Note that the structure represented by the following formula is hereinafter referred to as "ssH amino linker".

[0101]

Chemical formula

[0102] (2) Synthesis of N-terminal modified peptide The peptide was synthesized using a contract synthesis service (Gene Design Co., Ltd.). The amino acid sequences of the synthesized peptides are shown below. C-OVA8: CSIINFEKL (SEQ ID NO: 30) C-TRP2-9: CSVYDFFVWL (SEQ ID NO: 31) C-TRP2-8: CVYDFFVWL (SEQ ID NO: 32) C-gp100-9: CKVPRNQDWL (SEQ ID NO: 33) C-gp100-8: CVPRNQDWL (SEQ ID NO: 34) CM-TRP2-9: CMSVYDFFVWL (SEQ ID NO: 35) C-TRP2-13: CFANASVYDFFVWL (SEQ ID NO: 36) C-TRP2-11: CNASVYDFFVWL (SEQ ID NO: 37)

[0103] C-OVA8 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence at positions 258 to 265 of ovalbumin (OVA; GenBank accession number: CAA23716.1) (hereinafter also referred to as OVA peptide 1). C-TRP2-9 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence at positions 180 to 188 of mTRP2 (mouse Tyrosinase-related protein 2; GenBank accession number: CAA44951.1) (hereinafter also referred to as TRP2-9). C-TRP2-8 is a peptide consisting of a sequence obtained by adding cysteine to the N-terminus of the amino acid sequence at positions 181 to 188 of mTRP2. C-gp100-9 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence at positions 25 to 33 of hGP100 (human glycoprotein 100; GenBank accession number: AAC60634.1) (hereinafter also referred to as hGP100-9). C-gp100-8 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence at positions 26 to 33 of hGP100. CM-TRP2-9 is a peptide obtained by adding cysteine and methionine to the N-terminus of a peptide consisting of the amino acid sequence at positions 180 to 188 of mTRP2. C-TRP2-13 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence at positions 176 to 188 of mTRP2. C-TRP2-11 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence at positions 178 to 188 of mTRP2.

[0104] (3) Synthesis of CpG DNA(S)-peptide conjugate 1 mol of the SPDP-modified CpG DNA(S) synthesized in (1) and 25 mol of the peptide synthesized in (2) were mixed in an aqueous solution of 30% N,N-dimethylformamide (DMF), allowed to stand at 40 °C for 3 hours, and then the CpG DNA(S)-peptide conjugate was fractionated by HPLC under any of the following conditions (A) to (C). Table 2 shows the HPLC conditions and retention times used for the fractionation of each conjugate. <HPLC condition (A)> Under the following gradient conditions, solution A was 0.1 M hexafluoroisopropanol, 8 mM triethylamine (TEA), solution B was methanol, the column was X-Bridge C18 2.5 μm 4.6 * 75 mm (Waters Corporation) was used, HPLC was performed at a column temperature of 60 °C and a flow rate of 1 mL / min. 0 min A: 95% B: 5% ~20 min A: 70% B: 30% <HPLC condition (B)> Under the following gradient conditions, solution A was 0.1 M hexafluoroisopropanol, 8 mM triethylamine (TEA), solution B was methanol, the column was X-Bridge C18 2.5 μm 4.6 * 75 mm (Waters Corporation) was used, HPLC was performed at a column temperature of 60 °C and a flow rate of 1 mL / min. 0 min A: 95% B: 5% ~25 min A: 60% B: 40% <HPLC condition (C)> Under the following gradient conditions, solution A was 0.1 M triethylammonium acetate (TEAA; pH 7.0), solution B was acetonitrile, the column used was ZORBAX Eclipse Plus C18 (Agilent Technologies), the column temperature was 40 °C, and the flow rate was 1 mL / min for HPLC analysis. 0 min A: 90% B: 10% ~25 min A: 70% B: 30% ~30 min A: 0% B: 100% <HPLC Conditions (D)> Under the following gradient conditions, solution A was 0.1 M hexafluoroisopropanol and 8 mM triethylamine (TEA), solution B was methanol, the column used was X-Bridge C18 2.5 μm 4.6 * 75 mm (Waters Corporation), the column temperature was 60 °C, and the flow rate was 1 mL / min for HPLC analysis. 0 min A: 95% B: 5% ~25 min A: 50% B: 50%

[0105] In the fractionation of the solution after the reaction of SPDP-modified CpG DNA (S) with the peptide using HPLC, detection was performed by monitoring the absorption at 260 nm. It was confirmed that the elution time of the CpG DNA (S)-peptide conjugate after fractionation was slower than that of SPDP-modified CpG DNA (S). This is presumably because the elution time became slower due to binding to the hydrophobic peptide. Also, no peak of unreacted SPDP-modified CpG DNA (S) was observed in the chromatogram after fractionation, and only the peak of the CpG DNA (S)-peptide conjugate was detected, confirming that the target CpG DNA (S)-peptide conjugate was obtained with high purity. As an example of the results of HPLC analysis, Figure 1 shows the chromatogram of CpG30 (S)a-mTRP2pep9 (Compound 1) under the above condition (B).

[0106] The structure of the obtained CpG DNA(S)-peptide conjugate is shown below. In the formula, "CpG DNA" represents the base sequence portion of CpG30(S)a or CpG20(S)a, and "petide" represents the portion excluding the N-terminal cysteine of the peptide synthesized in (2) above.

[0107]

Chemical formula

[0108] The synthesized CpG DNA(S)-peptide conjugate is as follows.

[0109]

Table 2

[0110] Also shown in Figure 2 is an example of the mass spectrometry results, the mass spectrum (MALDI-TOF) of CpG30(S)a-mTRP2pep9 (Compound 1). Peaks of 11248.28 (monovalent negative ion) and 5622.53 (divalent negative ion) were detected, and it was confirmed that a mass spectrum corresponding to CpG30(S)a-mTRP2pep9 (theoretical molecular weight (theoretical most abundant mass for C 369 H 480 N 116 O 174 P 30 S 31 ) 11246.56) was obtained.

[0111] Example 2: Evaluation of cytotoxic T cell induction by CpG DNA(S)-peptide conjugate (evaluation using mTRP2 antigen) (1) Method for evaluating cytotoxic T cell induction CpG DNA(S)-peptide conjugate was administered intradermally to mice (C57BL / 6 mice (♂, 7 weeks old)). The dose of CpG DNA(S)-peptide conjugate was 50, 200, or 1000 ng in terms of peptide per mouse (in this case, approximately 0.4, 1.7, or 8.5 μg in terms of CpG30(S)). One week after administration, splenocytes were taken from non-administered individuals of the same strain of mice. These were divided into two groups of 2.0×10 7 cells / ml each. To one group, peptide was added to a concentration of 10 μg / mL as an antigen, and antigen-retaining splenocytes were prepared by allowing it to stand for 90 minutes. Splenocytes to which no peptide was added were used as antigen-non-retaining splenocytes. Both antigen-retaining splenocytes and antigen-non-retaining splenocytes were fluorescently modified using 5,6-carboxyfluorescein succinimidyl ester (CFSE). At this time, by changing the concentration of CFSE, the antigen-retaining splenocytes (CFSE: 1 μM) had a higher fluorescence intensity than the antigen-non-retaining splenocytes (CFSE: 0.1 μM). Equal numbers of administered antigen-retaining splenocytes and antigen-non-retaining splenocytes were mixed and intravenously administered to mouse individuals administered with CpG DNA(S)-peptide conjugate at a cell number of 3.0×10 6 via the tail vein one week after administration. Twenty-four hours after the above-mentioned intravenous administration via the tail vein, splenocytes were taken from the mice, and the ratio of antigen-retaining splenocytes to antigen-non-retaining splenocytes was quantified by flow cytometry. The activity of the induced antigen-specific cytotoxic T cells was evaluated by assessing the reduction in the amount of antigen-retaining splenocytes. For comparison, measurements were performed under the same conditions using mouse individuals administered with PBS (phosphate-buffered saline) instead of CpG DNA(S)-peptide conjugate as a control group.

[0112] (2) Evaluation of the cytotoxic T cell-inducing ability of CpG30(S)a-mTRP2pep10 In the prior application (PCT / JP2019 / 038090), it was shown by a test using a peptide having antigenicity derived from OVA that the CpG DNA(S)-peptide conjugate has high cytotoxic T lymphocyte (CTL) induction ability. On the other hand, it was found that when a similar CpG DNA(S)-peptide conjugate was prepared for other antigen peptides, sufficient CTL induction ability might not be obtained (Figure 3). Specifically, when evaluating the CTL induction ability of CpG30(S)a-OVApep9 using a peptide (OVA peptide 1) consisting of the amino acid sequence at positions 258 to 265 of ovalbumin (OVA), which is known as a peptide having antigenicity, disappearance of antigen-retaining splenocytes was observed at a dose of 20 ng in terms of peptide per mouse tail, and it was confirmed that strong CTL activity was induced. In contrast, when evaluating the CTL induction ability of CpG30(S)a-mTRP2pep10 (a conjugate prepared using a peptide with a cysteine added to the N-terminus of TRP2-9), a peptide consisting of the amino acid sequence (9 amino acids) at positions 180 to 188 of mTRP2, which is known as a melanoma-related antigen, no CTL activity was observed even at a dose of 200 ng in terms of peptide per mouse tail.

[0113] (3) Evaluation of cytotoxic T lymphocyte induction ability of CpG30(S)a-mTRP2pep9 In order for the CpG DNA(S)-peptide conjugate to induce CTL activity, after being taken up by antigen-presenting cells, the peptide moiety needs to be detached from the polynucleotide moiety and the spacer moiety and bind to MHC molecules. From the above results, it was considered that C-TRP2-9 (10 amino acids) used in the preparation of CpG30(S)a-mTRP2pep10, in which one amino acid is added to the N-terminus of the peptide having the original antigenicity, might not be able to bind to MHC molecules or, even if it binds, might not be recognized by the T cell receptor. Therefore, a peptide (C-TRP2-8) was prepared by adding cysteine after removing one residue (serine) at the N-terminus of TRP2-9 consisting of 9 amino acids, and CpG DNA(S)-peptide conjugate CpG30(S)a-mTRP2pep9 was prepared and its ability to induce cytotoxic T cells was evaluated. TRP2-9 was used as an antigen for preparing antigen-retaining splenocytes. The measurement results by flow cytometry are shown in Fig. 4. In CpG30(S)a-mTRP2pep9, a significant decrease in antigen-retaining splenocytes was observed at a dose of 200 ng in terms of peptide per mouse, and it was confirmed that high CTL activity was induced. In contrast, in CpG30(S)a-mTRP2pep10, almost no decrease in antigen-retaining splenocytes was observed.

[0114] (4) Dose-dependence The ability to induce cytotoxic T cells was evaluated with the dose of CpG30(S)a-mTRP2pep9 being 50, 200, or 1000 ng in terms of peptide per mouse. TRP2-9 was used as an antigen for preparing antigen-retaining splenocytes. The measurement results by flow cytometry are shown in Fig. 5. Dose-dependence was recognized in the CTL-inducing ability of CpG30(S)a-mTRP2pep9. It was shown that strong CTL activity could be induced at a dose of 200 ng.

[0115] (5) Base-length dependence of CpG DNA A CpG DNA(S)-peptide conjugate CpG20(S)a-mTRP2pep9 was prepared by substituting the polynucleotide part of CpG30(S)a-mTRP2pep9 with CpG20(S)a with a length of 20 bases, and its ability to induce cytotoxic T cells was evaluated. TRP2-9 was used as an antigen for preparing antigen-retaining splenocytes. The measurement results by flow cytometry are shown in Fig. 6. It was shown that CpG20(S)a-mTRP2pep9 has a high CTL-inducing ability similar to that of CpG30(S)a-mTRP2pep9.

[0116] Example 3: Evaluation of MHC-I binding of the peptide used in the CpG-peptide conjugate of the present invention (evaluation of MHC-I binding competition inhibition with OVA peptide 1 in DC2.4 cells) After detaching mouse dendritic cell line DC2.4 cells from the dish, suspend them in a 1.5 mL tube with 1.5×10 5 cells / 200 μL PBS, add OVA peptide 1 to a concentration of 0.25 μg / mL, and add a peptide derived from TRP2 (TRP2-9, C-TRP2-9, or C-TRP2-8) to a concentration of 2.5 μg / mL in terms of peptide. Incubate the tube in an ice bath for 30 minutes. Next, add an antibody specific to the molecular complex of OVA peptide 1 and MHC-I (fluorescently labeled with phycoerythrin (PE): PE-labeled anti-mouse OVA 257-264 (SIINFEKL) peptide bound to H-2Kb antibody (ThermoFisher SCIENTIFIC)) (hereinafter referred to as "PE labelled H-2Kb / FIINFEKL"). Quantify the OVA peptide 1-MHC-I complex bound by the antibody on DC2.4 cells by flow cytometry, and evaluate the MHC-I binding competition inhibition activity by the peptide derived from TRP2. The measurement results of flow cytometry are shown in Fig. 7. When TRP2-9 was added, the average fluorescence intensity decreased by 38%, indicating that TRP2-9 has an inhibitory effect on the binding of OVA peptide 1 to MHC class I molecules, that is, it can bind to MHC class I molecules. Also, when C-TRP2-8, which has the same amino acid length but different N-terminal amino acids from TRP2-9, was added, the average fluorescence intensity decreased by 23%, and an inhibitory effect on the binding of OVA peptide 1 to MHC class I molecules was confirmed. On the other hand, no inhibitory effect was observed with C-TRP2-9, which is one amino acid longer than TRP2-9. Therefore, it was suggested that the binding to MHC-1 was lost with C-TRP2-9 and maintained with C-TRP2-8.

[0117] Example 4: Evaluation of cytotoxic T cell induction by CpG DNA(S)-peptide conjugate (evaluation using hGP100) Using CpG30(S)a-hGP100pep10 or CpG30(S)a-hGP100pep9, the ability to induce cytotoxic T cells was evaluated in the same manner as in Example 2. As an antigen for preparing antigen-retaining splenocytes, a peptide (hGP100-9) consisting of the 25th to 33rd amino acid sequences (9 amino acids) of hGP100, which is known as a melanoma-related antigen, was used. The measurement results of flow cytometry are shown in Figs. 8 and 9. Similar to the case of evaluation using mTRP2, in CpG30(S)a-hGP100pep9, a decrease in antigen-retaining splenocytes was observed at a dose of 200 ng in terms of peptide per tail, and it was confirmed that CTL activity was induced. In contrast, in CpG30(S)a-hGP100pep10, almost no decrease in antigen-retaining splenocytes was observed (Fig. 8). Also, a dose-dependence was recognized in the CTL-inducing ability of CpG30(S)a-hGP100pep9, and it was shown that strong CTL activity could be induced at a dose of 200 ng (Fig. 9).

[0118] Reference Example 1: Evaluation of cytotoxic T cell induction by CpG30(S)a-CMTRP2-9 For the presentation of antigen peptides by MHC-1, endoplasmic reticulum aminopeptidase (ERAP) is involved. Antigen peptide precursors are trimmed from the N-terminus by ERAP to a length suitable for binding to MHC-1. Regarding this process, it has been reported that in antigen peptide precursors having a cysteine at the N-terminus, inserting alanine, leucine, or methionine on the C-terminal side of cysteine makes them more susceptible to trimming by ERAP (WO2014 / 157704). From the results shown in Fig. 3, it was considered that for CpG30(S)a-mTRP2pep10 as well, inserting alanine, leucine, or methionine on the C-terminal side of the cysteine at the N-terminus of C-TRP2-9 would make it more susceptible to trimming by ERAP and the CTL-inducing ability might be improved. Therefore, using CM-TRP2-9 in which methionine was inserted on the C-terminal side of the cysteine at the N-terminus of C-TRP2-9, the CpG DNA(S)-peptide conjugate CpG30(S)a-CMTRP2-9 was prepared, and its CTL-inducing ability was evaluated. TRP2-9 was used as the antigen for preparing antigen-retaining splenocytes. The measurement results by flow cytometry are shown in Fig. 10. A sufficient decrease in antigen-retaining splenocytes was not observed even at a dose of 1000 ng in terms of peptide per tail, and there was almost no decrease at a dose of 200 ng. In the in vitro evaluation using the peptide (CM-TRP2-9), results suggesting that it was more easily cleaved by ERAP were also obtained, but it was suggested that the CTL-inducing ability could not be sufficiently improved in vivo.

[0119] Reference Example 2: Evaluation of cytotoxic T cell induction by CpG30(S)a-mTRP2-14 and CpG30(S)a-mTRP2-12 The susceptibility to trimming by ERAP varies depending on the peptide chain length, and it has been reported that peptides 9 to 16 amino acids in length are preferred substrates for ERAP (Proc Natl Acad Sci U S A. 102(47):17107-12 (2005)). Therefore, using sequences C-TRP2-11 and C-TRP2-13 that are 2 or 4 amino acids longer at the N-terminal side than the sequence derived from TRP2 in C-TRP2-9, the CpG DNA(S)-peptide conjugates CpG30(S)a-TRP2-12 and CpG30(S)a-TRP2-14 were prepared, and their CTL-inducing abilities were evaluated. TRP2-9 was used as the antigen for preparing antigen-retaining splenocytes. The measurement results by flow cytometry are shown in Fig. 11. No decrease in antigen-retaining splenocytes was observed for any of the CpG DNA(S)-peptide conjugates. In the in vitro evaluation using the peptides (C-TRP2-11 or C-TRP2-13), results suggesting that they were more easily cleaved by ERAP were also obtained, but it was suggested that the CTL-inducing ability could not be sufficiently improved in the in vivo evaluation using the conjugates.

[0120] Example 5: Synthesis of CpG DNA(S)-peptide conjugate containing CpG motifs other than those derived from K3 and evaluation of its cytotoxic T cell-inducing ability (1) Synthesis method A CpG DNA(S)-peptide conjugate containing CpG motifs other than those derived from K3 was synthesized by the same method as in Example 1. The synthesized compounds are shown in the following formula and Table 3. As the N-terminal modified peptide, C-TRP2-8 was used (see Example 1(2)).

[0121]

Chemical formula

[0122]

Table 3

[0123] (2) Test method The cytotoxic T cell-inducing abilities of ISS1018-mTRP2pep9, ODN2006-mTRP2pep9, and ODN1826-mTRP2pep9 were evaluated by the same method as in Example 2. The dose was 200 ng in terms of peptide per fish. As a control, CpG30(S)a-mTRP2pep9 (conjugate containing CpG motifs derived from K3; see Example 1) was used. The results are shown in Figure 12. It was shown that all the compounds synthesized in (1) had the ability to induce cytotoxic T cells, similar to the conjugate containing CpG motifs derived from K3.

[0124] Example 6: Synthesis of a conjugate having an ssH amino linker moiety in which a phosphate group is substituted with a thiophosphate group and evaluation of its cytotoxic T cell-inducing ability (1) Synthesis method A conjugate having an ssH amino linker moiety in which a phosphate group is substituted with a thiophosphate group was synthesized by the following method. The synthesized compounds are shown in the following formula and Table 4.

[0125] [Chemistry]

[0126] [Table 4]

[0127] (1-1) Synthesis of CpG DNA(S) derivatives The synthesis of CpG DNA(S) was carried out using the phosphoramidite method (e.g., Nucleic Acids Research, 12, 4539 (1984)). The synthesis of CpG DNA(S) with amino group modification was carried out using a ssH Amino Linker (Bioorg. Med. Chem., 16, 941-949 (2008)) in which the phosphate group was replaced with a thiophosphate group. For these syntheses, a contract synthesis service (GeneDesign Inc.) was utilized. The nucleotide sequence of the synthesized CpG DNA(S) is the same as that of CpG30a described in Example 1(1). The obtained amino group-modified CpG DNA(S) has a structure represented by the following formula at the 5'-end. Hereinafter, among the CpG DNA(S) derivatives having a structure represented by the following formula at the 5'-end, those having the sequence of SEQ ID NO: 6 are referred to as "CpG30(S)a2".

[0128] [Chemistry]

[0129] Furthermore, 1 mol of amino group-modified CpG DNA(S) and 30 mol of succinimidyl 6-[3'-(2-pyridyldithio)-propionamido]hexanoate (LC-SPDP) were mixed in a phosphate buffer (pH 8.0), allowed to stand at 40 °C for 3 hours, and then the SPDP-modified CpG DNA(S) was purified using a NAP-5 column. The obtained SPDP-modified CpG DNA(S) has a structure represented by the following formula.

[0130] [Chemical formula]

[0131] (1-2) Synthesis of N-terminal modified peptide The peptide was synthesized using a contract synthesis service (Gene Design Co., Ltd.). The amino acid sequences of the synthesized peptides are shown below. C-OVA7: CIINFEKL (SEQ ID NO: 46) C-TRP2-8: CVYDFFVWL (SEQ ID NO: 32)

[0132] C-OVA7 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence at positions 259-265 of ovalbumin (OVA; GenBank accession number: CAA23716.1). C-OVA7 is a peptide obtained by deleting the N-terminal 1 residue of OVA peptide 1 (a peptide consisting of the amino acid sequence of SEQ ID NO: 41), which is an MHC-1 binding peptide, and adding cysteine to the N-terminus. C-TRP2-8 is as described in Example 1, and is a peptide obtained by deleting the N-terminal 1 residue of TRP2-9, which is an MHC-1 binding peptide, and adding cysteine to the N-terminus.

[0133] (1-3) Synthesis of CpG DNA(S)-peptide conjugate To the SPDP-modified CpG DNA(S) synthesized in (1-1), 5 to 10 molar equivalents of the peptide synthesized in (1-2) were mixed in an aqueous solution of 50% dimethyl sulfoxide (DMSO) and reacted at 40°C for 2 hours. After the reaction, the CpG DNA(S)-peptide conjugate was separated by HPLC purification under the following conditions. <HPLC conditions (E)> Under the following gradient conditions, Solution A was 0.1 M hexafluoroisopropanol (HFIP), 8 mM triethylamine (TEA), Solution B was methanol, the column used was XBridge BEH C18 (4.6×75 mm Column XP) (Waters Corporation), the column temperature was 40 °C, and the flow rate was 1 mL / min for HPLC analysis. 0 min A: 80% B: 20% ~10 min A: 50% B: 50% Table 4 shows the HPLC conditions and retention times used for the fractionation of each conjugate.

[0134] (2) Test method In the same manner as in Example 2, the cytotoxic T cell-inducing ability of CpG30(S)a2-OVApep8 and CpG30(S)a2-mTRP2pep9 was evaluated. The dosage of CpG30(S)a2-OVApep8 was 20 ng in terms of peptide per fish. The dosage of CpG30(S)a2-mTRP2pep9 was 200 ng in terms of peptide per fish. The difference in dosage between the two conjugates reflects the difference in the antigenicity strength of the original peptides themselves. The results are shown in Figure 13. It was shown that both CpG30(S)a2-OVApep8 and CpG30(S)a2-mTRP2pep9 synthesized in (1) have high cytotoxic T cell-inducing ability.

[0135] Example 7: Evaluation of cytotoxic T cell induction by CpG DNA(S)-peptide conjugate (activity in two administrations) The cytotoxic T cell-inducing ability when CpG30(S)a-mTRP2pep9 was administered twice was evaluated in the same manner as in Example 2. The second administration was carried out 10 days after the first administration. The dosage was 200 ng in terms of peptide per fish for both administrations. The results are shown in Figure 14. It was suggested that by administering the CpG DNA(S)-peptide conjugate twice, the CTL activity was improved compared to the case of single administration.

[0136] Example 8: Synthesis of double-stranded CpG DNA(S)-peptide conjugate A double-stranded complex (double-stranded CpG DNA(S)-peptide conjugate) was prepared by annealing the CpG DNA(S)-peptide conjugate (CpG30(S)a-mTRP2pep9; see Example 1) with a DNA derivative having a sequence complementary to the base sequence of the CpG DNA(S) portion thereof. (1) Synthesis of CpG complementary strand DNA derivative The synthesis of DNA complementary to CpG was carried out using the phosphoramidite method in the same manner as for CpG DNA(S). The synthesis of complementary strand DNA having a lipid modification at the 5'-end was carried out by using the lipidated phosphoramidite represented by the following formula (synthesized in the same manner as M22-12 phosphoramidite in the literature (WO2017 / 057540)) in the final coupling of the sequence synthesis of the complementary strand DNA.

[0137]

Chemical formula

[0138] The synthesis of complementary strand DNA having a lipid modification at the 3'-end was carried out as follows. On a universal solid support (Glen UnySupport 500 (GlenResearch, 20-5040)), after performing the first coupling using Asymmetric Doubler(Lev) Phosphoramidite (GlenResearch, 10-1981), the sequence of the complementary strand DNA was synthesized. Then, after detritylation and acetyl protection on the solid support, the levulinic acid unit was deprotected by the method specified by the manufacturer. The target compound was synthesized by reacting the resulting hydroxyl group with the above lipidated phosphoramidite.

[0139] The synthesis of complementary strand DNA having lipid modifications at both the 3' and 5'-ends was carried out as follows. After the first coupling was performed using Asymmetric Doubler (Lev) Phosphoramidite (GlenResearch, 10-1981) on a universal solid support (Glen UnySupport 500 (GlenResearch, 20-5040)), the sequence of the complementary strand DNA was synthesized. Subsequently, after detritylation was performed on the solid support, the levulinic acid unit was deprotected by the method specified by the manufacturer. The target compound was synthesized by reacting the above lipidated phosphoramidite with the resulting 3’ and 5’-terminal hydroxyl groups.

[0140] The structure of the DNA derivative complementary to the synthesized CpG is shown below. The DNA sequence (compK3: SEQ ID NO: 47) in the following derivative is a sequence complementary to the base sequence of K3 (SEQ ID NO: 1). 5'-Lipo-compK3: 5'-Lipo^G^A^G^AACGCTCGAGA^G^T-3' 3'-Lipo-compK3: 5'-G^A^G^AACGCTCGAGA^G^T^Lipo-3' 5',3'-di-Lipo-compK3: 5'-Lipo^G^A^G^AACGCTCGAGA^G^T^Lipo-3'

[0141] In the above structure, " ^ " indicates a phosphorothioate bond between nucleosides. Also, "Lipo^G" and "T^Lipo" represent the following structures.

[0142]

Chemical formula

[0143] The DNA derivative complementary to CpG was fractionated by HPLC under the following conditions. The results are shown in Table 5. <HPLC conditions (F)> Under the following gradient conditions, Solution A was 0.1 M hexafluoroisopropanol (HFIP), 8 mM triethylamine (TEA), Solution B was methanol, the column used was Clarity (registered trademark) 2.6 μm Oligo-MS 100A (LC-Column 50 x 2.1 mm) (Phenomenex Inc), the column temperature was 60 °C, and the flow rate was 0.5 mL / min. HPLC was performed. 0 min A: 90% B: 10% ~7 min A: 10% B: 90%

[0144]

Table 5

[0145] (2) Formation of double-stranded complex with CpG DNA(S)-peptide conjugate The PBS solution of CpG30(S)a-mTRP2pep9 and the PBS solution of the lipid-modified compK3 synthesized in (1) above were mixed in a 1.5 mL tube so that the final concentration was 3.4 μM each. After heating this by placing it in a hot water bath at 90 °C and then leaving it overnight to gradually return to room temperature (double strands are formed during this cooling process). When 50 μL of this solution is administered to a mouse, it is equivalent to administering 200 ng of the peptide. Five types of double-stranded complexes of the combinations described in Table 6 below were obtained. The formation of the double-stranded complex can be confirmed by electrophoresis using polyacrylamide gel or the like, liquid chromatography such as size exclusion chromatography, measurement of the melting temperature using ultraviolet spectroscopy, measurement of the aggregate molecular weight by static light scattering method, etc.

[0146]

Table 6

[0147] Example 9: Synthesis of CpG DNA(S)-peptide conjugate using MHC-2 binding peptide and evaluation of CD4 + T cell activation (1) Synthesis method By the following method, two conjugate CpGs, CpG30(S)a2-OVA2-15 and CpG30(S)a2-OVA2-17, having a CpG DNA(S) moiety derived from an MHC-2 binding peptide were synthesized. The synthesized compounds are shown in the following formula and Table 7.

[0148]

Chemical formula

[0149]

Table 7

[0150] (1-1) Synthesis of CpG DNA(S) derivative By the same method as in Example 6, CpG30(S)a2 and its SPDP-modified product were synthesized. (1-2) Synthesis of N-terminal modified peptide The N-terminal cysteine-modified peptide was synthesized by a general Fmoc solid-phase peptide synthesis method. The amino acid sequences of the synthesized peptides are shown below. The amino acid sequences of the synthesized peptides are shown below. C-OVA2-14: CSQAVHAAHAEINEA (SEQ ID NO: 48) C-OVA2-16: CSQAVHAAHAEINEAGR (SEQ ID NO: 51)

[0151] C-OVA2-14 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence at positions 325-338 of ovalbumin (OVA; GenBank accession number: CAA23716.1). C-OVA2-14 is a peptide obtained by deleting the N-terminal 1 residue and the C-terminal 2 residues of OVA peptide 2 (a peptide consisting of the amino acid sequence of SEQ ID NO: 42 containing the mouse I-Ab / I-Ad binding sequence) and adding cysteine to the N-terminus. C-OVA2-16 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence 325-340 of the ovalbumin. C-OVA2-16 is a peptide obtained by deleting the N-terminal 1 residue of the OVA peptide 2 and adding cysteine to the N-terminus.

[0152] (1-3) Synthesis of CpG DNA(S)-peptide conjugate Using the SPDP-modified CpG DNA(S) synthesized in (1-1) and the peptide synthesized in (1-2), a CpG DNA(S)-peptide conjugate was synthesized in the same manner as (1-3) of Example 6.

[0153] (2) Test method (Evaluation of CD4 + T cell activation by single immunization with CpG-MHC2 peptide conjugate by measuring IFN-γ secretion activity) CpG DNA(S)-peptide conjugate was administered intradermally to mice (C57BL / 6 mice (♂, 7 weeks old)) as an antigen. The dose of CpG DNA(S)-peptide conjugate was 1000 ng in terms of peptide per mouse. One week after administration, splenocytes were taken out and seeded in a 96-well dish at 1.0×10 6 cells / 100 μL (medium; RPMI1640), and an MHC-2-binding antigen peptide derived from OVA (OVA 324-340 : ISQAVHAAHAEINEAGR (SEQ ID NO: 42)) was added to a concentration of 10 μg / mL. After 24 hours, interferon γ (IFN-γ) in the medium was quantified using an IFN gamma Mouse ELISA Kit (Invitrogen, IFN gamma 'Femto-HS' High Sensitivity Mouse Uncoated ELISA Kit). When mice were immunized by administration of CpG DNA(S)-peptide conjugate, antigen-specific CD4 + T cells in splenocytes were activated by stimulation with the addition of the antigen peptide to the medium and secreted IFN-γ.

[0154] The results are shown in Figures 15 and 17. In the splenocytes of mice administered with CpG30(S)a2-OVA2-15 and CpG30(S)a2-OVA2-17, respectively, high IFN-γ secretion activities were observed in both (Figs. 15 and 17; see Reference Example 3 for CpG30(S)a2-OVA2-18). When conjugates are prepared using MHC-2-binding peptides, it was shown that the C-terminal peptide may be deleted.

[0155] Reference Example 3: Synthesis of CpG DNA(S)-peptide conjugates using MHC-2-binding peptides and evaluation of CD4 + T cell activation (1) Synthesis method Two types of conjugates, CpG30(S)a2-OVA2-18 and CpG30(S)a2-OVA2-18c, having a CpG DNA(S) moiety derived from an MHC-2-binding peptide were synthesized by the following method. The synthesized compounds are shown in the following formula and Table 8.

[0156]

Chemical formula

[0157]

Table 8

[0158] (1-1) Synthesis of CpG DNA(S) derivatives CpG30(S)a2 and its SPDP-modified product were synthesized by the same method as in Example 6. (1-2) Synthesis of N-terminal modified peptides and C-terminal modified peptides N-terminal cysteine-modified peptides and C-terminal cysteine-modified peptides were synthesized by a general Fmoc solid-phase peptide synthesis method. The amino acid sequences of the synthesized peptides are shown below. C-OVA2-17: CISQAVHAAHAEINEAGR (SEQ ID NO: 49) OVA2-17-C: ISQAVHAAHAEINEAGRC (SEQ ID NO: 50)

[0159] C-OVA2-17 is a peptide obtained by adding cysteine to the N-terminus of a peptide consisting of the amino acid sequence 324-340 of ovalbumin. C-OVA2-17 is a peptide obtained by adding cysteine to the N-terminus of the said OVA peptide 2. OVA2-17-C is a peptide obtained by adding cysteine to the C-terminus of a peptide consisting of the amino acid sequence 324-340 of ovalbumin. C-OVA2-17 is a peptide obtained by adding cysteine to the C-terminus of the said OVA peptide 2.

[0160] (1-3) Synthesis of CpG DNA(S)-peptide conjugate Using the SPDP-modified CpG DNA(S) synthesized in (1-1) and the peptide synthesized in (1-2), a CpG DNA(S)-peptide conjugate was synthesized in the same manner as in (1-3) of Example 6.

[0161] (2) Test method (Evaluation of CD4 + T cell activation by a single immunization with CpG-MHC2 peptide conjugate by measuring IFN-γ secretion activity) The CpG DNA(S)-peptide conjugate was administered intradermally to mice (C57BL / 6 mice (♂, 7 weeks old)) as an antigen. The dose of the CpG DNA(S)-peptide conjugate was 1000 ng in terms of peptide per mouse. One week after the administration, splenocytes were removed and seeded in a 96-well dish at 1.0×10 6 cells / 100 μL (medium; RPMI1640), and an antigen peptide derived from OVA (OVA 324-340:ISQAVHAAHAEINEAGR (SEQ ID NO: 42) was added to a concentration of 10 μg / mL. After 24 hours, interferon γ (IFN-γ) in the medium was quantified using an IFN gamma Mouse ELISA Kit (Invitrogen, IFN gamma 'Femto-HS' High Sensitivity Mouse Uncoated ELISA Kit). When mice were immunized by administration of CpG DNA(S)-peptide conjugate, stimulation by addition of the antigen peptide into the medium activated antigen-specific CD4 + T cells, which secrete IFN-γ.

[0162] The results are shown in FIGS. 15 and 16. High IFN-γ secretion activity was observed in splenocytes of mice administered CpG30(S)a2-OVA2-18 (FIGS. 15 and 16). Also, activity equivalent to or higher than that in the case of administration of CpG30(S)a2-OVA2-18 was observed in splenocytes of mice administered CpG30(S)a2-OVA2-18c (FIG. 16). It was shown that when conjugates are prepared using MHC-2 binding peptides, CpG DNA(S) may be conjugated to the C-terminal side.

[0163] Example 10: Preparation of CpG DNA(S)-peptide conjugate (2) and evaluation of its cytotoxic T cell inducing ability (1) Synthesis method (1-1) Synthesis of CpG DNA(S) derivative Synthesis of CpG DNA(S) having a 6-mercaptohexyl group at the 5'-end was performed by synthesizing the sequence of CpG DNA(S) using the phosphoramidite method and then reacting it with 5'-thiol-modifier C6 (S-Trityl-6-mercaptohexyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite). For these syntheses, a contract synthesis service (GeneDesign Co., Ltd.) was utilized. The obtained 6-mercaptohexyl-modified CpG DNA(S) has a structure represented by the following formula at the 5'-end. Hereinafter, among the CpG DNA(S) derivatives having a structure represented by the following formula at the 5'-end, those having the sequence of SEQ ID NO: 6 are referred to as "CpG30(S)a3".

[0164]

Chemical formula

[0165] In addition, the synthesis of the CpG DNA(S) derivative having a structure represented by the following formula at the 5'-end was carried out by reacting the amino group-modified CpG DNA(S) synthesized in (1-1) of Example 6 with PPC-NHS ester (2,5-Dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)butanoate) at a molar ratio of 1:30 in a phosphate buffer (pH 8.0) at 40 °C for 3 hours, and then purifying with a NAP-5 column.

[0166]

Chemical formula

[0167] (1-2) Synthesis of N-terminal modified peptide In the same manner as in Example 1, C-TRP2-8: CVYDFFVWL (SEQ ID NO: 32) was synthesized.

[0168] (1-3) Synthesis of CpG DNA(S)-peptide conjugate To the CpG30(S)a3 synthesized in (1-1), 30 molar equivalents of Npys-OMe (CAS: 68118-08-1) were mixed in an aqueous solution of 33% dimethyl sulfoxide (DMSO) and reacted overnight. Next, 5 molar equivalents of the peptide synthesized in (1-2) were mixed in an aqueous solution of 50% dimethyl sulfoxide (DMSO) and reacted at 40 °C for 2 hours. After the reaction, the CpG DNA(S)-peptide conjugate was separated by HPLC purification to obtain CpG30(S)a3-mTRP2pep9. Furthermore, with respect to the PPC-NHS ester-modified CpG DNA (S) synthesized in (1-1), 5 molar equivalents of the peptide synthesized in (1-2) were mixed in an aqueous solution of 50% dimethyl sulfoxide (DMSO) and reacted at 40 °C for 2 hours. After the reaction, the CpG DNA (S)-peptide conjugate was separated by HPLC purification to obtain CpG30(S)a2-MeS-mTRP2pep9.

[0169] The synthesized compounds are shown in the following formula and Table 9.

Chemical formula

[0170]

Table 9

[0171] (2) Test method In the same manner as in Example 2, the ability of CpG30(S)a3-mTRP2pep9 to induce cytotoxic T cells was evaluated. The dosage was 200 ng in terms of peptide per fish. As a control, CpG30(S)a2-mTRP2pep9 (a conjugate containing a CpG motif derived from K3; see Example 6) was used. The results are shown in Figure 18. CpG30(S)a3-mTRP2pep9 was shown to have the ability to induce cytotoxic T cells, similar to CpG30(S)a2-mTRP2pep9 with a different spacer structure.

[0172] Example 11: Synthesis of CpG DNA (S)-peptide conjugate containing a cysteine analog (1) Synthesis method Using a peptide with an N-terminal modified by a cysteine analog instead of cysteine, a CpG DNA (S)-peptide conjugate was synthesized in the same manner as in Example 1. The synthesized compounds are shown in the following formula and Table 10. As the N-terminal modified peptide, the following synthetic peptide in which the N-terminal amino acid is a non-natural amino acid was used. dC-mTRP2pep8: D-cysteine-VYDFFVWL (SEQ ID NO: 52) homoC-mTRP2pep8: L-homocysteine-VYDFFVWL (SEQ ID NO: 53) Pen-mTRP2pep8: L-penicillamine-VYDFFVWL (SEQ ID NO: 54)

[0173]

Chem.

[0174]

Table 10

[0175] (2) Test method In the same manner as in Example 2, the ability of CpG30(S)a2-Pen-mTRP2pep8 to induce cytotoxic T cells was evaluated. The dosage was 200 ng in terms of peptide per fish. As a control, CpG30(S)a2-mTRP2pep9 (a conjugate containing a CpG motif derived from K3; see Example 6) was used. The results are shown in Fig. 18. CpG30(S)a2-Pen-mTRP2pep8 containing a cysteine analog was shown to have the ability to induce cytotoxic T cells, similar to CpG30(S)a2-mTRP2pep9.

[0176] Example 12: Synthesis of CpG DNA(S)-peptide conjugate having peptides at both 5' and 3' ends of CpG DNA (1) Synthesis method Using the following methods (1-1) to (1-3), a CpG DNA(S)-peptide conjugate having peptides at both ends of CpG DNA was synthesized. The synthesized compounds are shown in the following formula and Table 11. As the N-terminal modified peptide, C-TRP2-8 was used (see Example 1(2)).

[0177]

Chem.

[0178]

Table 11

[0179] (1-1) Synthesis of CpG DNA(S) derivative For the synthesis of CpG DNA(S) with Amino Linker attached to both 5' and 3' ends, after synthesizing the CpG sequence using 3'-Amino-Modifier C6-dC CPG (Link Technologies Ltd.) by the phosphoramidite method, the ssH Amino Linker was reacted at the 5' end. These syntheses utilized a contract synthesis service (Gene Design Co., Ltd.). The nucleotide sequence of the synthesized CpG DNA(S) is the same as CpG30a described in Example 1(1). The obtained amino group-modified CpG DNA(S) has a structure represented by the following formula at the 5' end

Chemical formula

Chemical formula

[0180] Hereinafter, among the CpG DNA(S) derivatives having the structures represented by the above formulas at the 3' and 5' ends, the one having the sequence of SEQ ID NO: 6 is referred to as "CpG30(S)a4". Furthermore, CpG30(S)a4 and N-succinimidyl 6-[3'-(2-pyridyldithio)-propionamido] hexanoate (LC-SPDP) were mixed at a molar ratio of 1:30 in phosphate buffer (pH 8.0), allowed to stand at 40 °C for 3 hours, and then SPDP-modified CpG DNA(S)a4 was purified using a NAP-5 column.

[0181] (1-2) Synthesis of N-terminal modified peptide Similar to Example 1, C-TRP2-8: CVYDFFVWL (SEQ ID NO: 32) was synthesized.

[0182] (1-3) Synthesis of CpG DNA(S)-peptide conjugate For the SPDP-modified CpG DNA(S)a4 synthesized in (1-1), the peptide synthesized in (1-2) was mixed in an amount of 5 to 10 molar equivalents in an aqueous solution of 50% dimethyl sulfoxide (DMSO) and reacted at 40°C for 2 hours. After the reaction, the CpG DNA(S)-peptide conjugate was separated by HPLC purification. The HPLC conditions and retention times used for the separation are shown in Table 11. [Industrial Applicability]

[0183] According to the present invention, it is possible to provide an immunopotentiator capable of inducing CTL activity for a wide range of antigenic peptides, and a pharmaceutical composition containing the same. [Sequence Listing Free-Text]

[0184] SEQ ID NO: 1: K3 SEQ ID NO: 2: K3-20(b) SEQ ID NO: 3: K3-21 SEQ ID NO: 4: K3-24 SEQ ID NO: 5: K3-27 SEQ ID NO: 6: K3-30(a) SEQ ID NO: 7: K3-30(b) SEQ ID NO: 8: K3-40 SEQ ID NO: 9: K3-30(c) SEQ ID NO: 10: K3-30(d) SEQ ID NO: 11: K3-30(e) SEQ ID NO: 12: K3-30(f) SEQ ID NO: 13: K3-26(a) SEQ ID NO: 14: K3-26(b) SEQ ID NO: 15: ODN1668 SEQ ID NO: 16: ODN1668-30 SEQ ID NO: 17: ODN1668-40 SEQ ID NO: 18: ODN1826 Sequence number 19: ODN1826-30 Sequence number 20: ODN1826-40 Sequence number 21: ODN2006 Sequence number 22: ODN2006-30 Sequence number 23: ODN2006-40 Sequence number 24: ODN684 Sequence number 25: ODN684-30 Sequence number 26: ODN684-40 Sequence number 27: ODN D-SL01 Sequence number 28: ODN D-SL01-35 Sequence number 29: C-CpG#1 Sequence number 30: C-OVA8 Sequence number 31: C-TRP2-9 Sequence number 32: C-TRP2-8 Sequence number 33: C-gp100-9 Sequence number 34: C-gp100-8 Sequence number 35: CM-TRP2-9 Sequence number 36: C-TRP2-13 Sequence number 37: C-TRP2-11 Sequence number 38: C-OVA2-17 Sequence number 39: C-OVA2-14 Sequence number 40: C-OVA2-11 Sequence number 41: OVA peptide 1 Sequence number 42: OVA peptide 2 Sequence number 43: 1018ISS Sequence number 44: 1018ISS#30 Sequence number 45: 1018ISS#40 Sequence number 46: C-OVA7 Sequence number 47: compK3 Sequence number 48: C-OVA2-14 Sequence number 49: C-OVA2-17 Sequence number 50: OVA2-17-C Sequence number 51: C-OVA2-16 Sequence number 52: dC-mTRP2pep8; The first amino acid is D-cysteine. Accession number 53: homoC-mTRP2pep8; the first amino acid is L-homocysteine. Accession number 54: Pen-mTRP2pep8; the first amino acid is L-penicillamine.

Claims

1. An immunopotentiator comprising a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the polynucleotide-peptide conjugate consists of a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, a peptide, and a spacer covalently bonded to the polynucleotide or polynucleotide derivative at one end and covalently bonded to the peptide at the other end, the polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bonds are substituted with phosphorothioate bonds, the peptide is a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are substituted with amino acids having a reactive functional group for forming a covalent bond with the spacer, wherein the one or more consecutive amino acids do not include an anchor residue for MHC binding, the immunopotentiator.

2. The immunopotentiator according to claim 1, wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide are covalent bonds cleavable in a biological environment.

3. The immunopotentiator according to claim 1 or 2, wherein the amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an amino acid having a thiol in its side chain.

4. The immunopotentiator according to any one of claims 1 to 3, wherein the covalent bond between the spacer and the peptide is a disulfide bond.

5. The immunopotentiator according to any one of claims 1 to 4, wherein the MHC-binding peptide is an MHC-I-binding peptide.

6. The immunopotentiator according to claim 5, wherein the MHC-I-binding peptide is an HLA-A-binding peptide or an HLA-B-binding peptide.

7. The immunopotentiator according to claim 5 or 6, wherein the amino acid length of the MHC-I-binding peptide is 8 or more and 11 or less.

8. The immunopotentiator according to any one of claims 1 to 4, wherein the MHC-binding peptide is an MHC-II-binding peptide.

9. The immunopotentiator according to any one of claims 1 to 8, wherein the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs.

10. The immunopotentiator according to any one of claims 1 to 9, wherein the base length of the polynucleotide or polynucleotide derivative is 15 or more and 40 or less.

11. The immunopotentiator according to claim 10, wherein the base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less.

12. In the polynucleotide derivative in which at least a part of the phosphodiester bonds are substituted with phosphorothioate bonds, 50% or more of the phosphodiester bonds are substituted with phosphorothioate bonds. The immunopotentiator according to any one of claims 1 to 11.

13. In the polynucleotide derivative in which at least a part of the phosphodiester bonds are substituted with phosphorothioate bonds, 90% or more of the phosphodiester bonds are substituted with phosphorothioate bonds. The immunopotentiator according to claim 12.

14. The immunopotentiator according to any one of claims 1 to 13, wherein the spacer contains a repeating unit represented by the following formula. 【Chemical 1】 In the above formula, X represents an oxygen atom or a sulfur atom (where each X may be the same or different), R represents any one of (CH ２ ), (CH ｐ ), (CH ２ ), (CH ｑ ), (CH ２ CH ２ O), (CH ｍ (where m, p and q each independently represent a natural number of 10 or less). n represents a natural number of 10 or less.

15. The immunopotentiator according to any one of claims 1 to 14, wherein the spacer has a structure represented by any of the following formulas. 【Chemical Formula 2】

16. The immunopotentiator according to any one of claims 1 to 13, wherein the spacer has a structure represented by any of the following formulas. 【Chemical 3】

17. An immunopotentiator containing, as an active ingredient, the polynucleotide-peptide conjugate described in claim 1 or a pharmaceutically acceptable salt thereof, wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide are covalent bonds cleavable in a biological environment, and the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) derivative containing two or more CpG motifs. The polynucleotide or polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bond is replaced with a phosphorothioate bond. The immune inducer.

18. An immune inducer comprising, as an active ingredient, a polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to claim 1, The amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an amino acid having a thiol in another side chain, The covalent bond between the spacer and the peptide is a disulfide bond, The MHC-binding peptide is an MHC-I binding peptide, The MHC-I binding peptide is an HLA-A binding peptide or an HLA-B binding peptide, The amino acid length of the MHC-I binding peptide is 8 or more and 11 or less, The polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs, The base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less, In the polynucleotide derivative in which at least a part of the phosphodiester bond is replaced with a phosphorothioate bond, 90% or more of the phosphodiester bond is replaced with a phosphorothioate bond, The spacer has a structure represented by any of the following formulas, The immune inducer. 【Chemical 4】

19. The immune inducer according to any one of claims 1 to 18, further comprising a substance having immune activating activity as an adjuvant.

20. A pharmaceutical composition comprising the immune inducer according to any one of claims 1 to 19.

21. The pharmaceutical composition according to claim 20, which is for the treatment or prevention of an infectious disease, a tumor, or an allergic disease.

22. The pharmaceutical composition according to claim 20, which is for the treatment or prevention of a tumor.

23. The immune inducer according to any one of claims 1 to 19 for use in the treatment or prevention of an infectious disease, a tumor, or an allergic disease.

24. The immune inducer according to any one of claims 1 to 19 for use in the treatment or prevention of a tumor.

25. Use of the immune inducer according to any one of claims 1 to 19 for the manufacture of a medicament for the treatment or prevention of an infectious disease, a tumor, or an allergic disease.

26. Use of the immunomodulator according to any one of claims 1 to 19 for the manufacture of a medicament for the treatment or prevention of tumors.

27. A polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof, wherein the polynucleotide-peptide conjugate consists of a single-stranded polynucleotide or polynucleotide derivative containing a CpG motif, a peptide, and a spacer covalently bound to the polynucleotide or polynucleotide derivative at one end and covalently bound to the peptide at the other end, wherein the polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bond is replaced by a phosphorothioate bond, wherein the peptide is a peptide in which one or more consecutive amino acids at the N-terminus of the MHC-binding peptide are replaced by amino acids having a reactive functional group for forming a covalent bond with the spacer, and wherein the one or more consecutive amino acids do not include an anchor residue for MHC binding, the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof.

28. The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to claim 27, wherein one or both of the covalent bond between the spacer and the polynucleotide or polynucleotide derivative and the covalent bond between the spacer and the peptide are covalent bonds cleavable in a biological environment, wherein the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) derivative containing two or more CpG motifs, wherein the polynucleotide or polynucleotide derivative is a polynucleotide derivative in which at least a part of the phosphodiester bond is replaced by a phosphorothioate bond, the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof.

29. The polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof according to claim 27, wherein the amino acid having a reactive functional group for forming a covalent bond with the spacer is cysteine or an amino acid having a thiol in its side chain, wherein the covalent bond between the spacer and the peptide is a disulfide bond, wherein the MHC-binding peptide is an MHC-I binding peptide, The MHC-I binding peptide is an HLA-A binding peptide or an HLA-B binding peptide, the amino acid length of the MHC-I binding peptide is 8 or more and 11 or less, the polynucleotide or polynucleotide derivative is a polydeoxyribonucleotide (DNA) or DNA derivative containing two or more CpG motifs, the base length of the polynucleotide or polynucleotide derivative is 20 or more and 30 or less, in the polynucleotide derivative in which at least a part of the phosphodiester bond is replaced with a phosphorothioate bond, 90% or more of the phosphodiester bond is replaced with a phosphorothioate bond, the spacer has a structure represented by any of the following formulas, the polynucleotide-peptide conjugate or a pharmaceutically acceptable salt thereof. 【Chemical Formula 5】

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