Immunogenic carrier and immunogenic composition

Abstract

Provided is an immunogenic carrier comprising a protein and an amino polysaccharide that contains 100 or more amino groups per molecule. Also provided is an immunogenic composition comprising the immunogenic carrier and a short-chain peptide.

Description

Immunogenic carriers and immunogenic compositions

[0001] The present invention relates to an immunogenic carrier and an immunogenic composition. This application claims priority to Japanese Patent Application No. 2023-173700, filed on October 5, 2023, the contents of which are incorporated herein by reference.

[0002] Antibody drugs targeting immune checkpoint molecules such as PD-L1 have dramatically changed the treatment of cancer, and cancer immunotherapy has become widely used in developed countries. As an alternative to antibody drugs directed against target molecules, therapeutic vaccines in which target molecules or portions thereof are bound to highly immunogenic carriers are being developed. Administration of these therapeutic vaccines is expected to induce antibody production against the target molecules, achieving similar effects to those of antibody drugs. A therapeutic vaccine that induces antibody production is required to break self-tolerance and induce antibody production against self-molecules. Therefore, a highly immunogenic carrier is required. Substances such as virus-like particles (VLPs) and keyhole limpet hemocyanin (KLH) have been used as such carriers (see, for example, Patent Document 1).

[0003] Special Publication No. 2008-539271

[0004] Antibody drugs are expensive because they must be produced in cultured animal cells, placing a heavy burden on the medical economy and, in many countries, on patients. VLPs, which are used as highly immunogenic carriers, must be constructed from multiple components, such as in bacteria, and KLH must be purified from shellfish. Therefore, the production of VLPs and KLH is not easy and is expensive. Furthermore, to produce safe therapeutic vaccines, it is necessary to minimize peptides derived from target molecules. To produce safe therapeutic vaccines, appropriate peptide design and highly immunogenic carriers are required.

[0005] Therefore, an object of the present invention is to provide an immunogenic carrier that is highly immunogenic, inexpensive, and highly safe, and an immunogenic composition that uses the immunogenic carrier.

[0006] The present invention includes the following aspects: [1] An immunogenic carrier comprising an aminopolysaccharide containing 100 or more amino groups per molecule and a protein. [2] The immunogenic carrier according to [1], wherein the aminopolysaccharide and the protein are chemically bonded. [3] The immunogenic carrier according to [1] or [2], wherein the aminopolysaccharide and the protein are not chemically bonded. [4] The immunogenic carrier according to any one of [1] to [3], wherein the protein is a bacterial toxoid. [5] The immunogenic carrier according to [4], wherein the bacterial toxoid is tetanus toxoid. [6] The immunogenic carrier according to any one of [1] to [5], wherein the aminopolysaccharide is aminodextran. [7] An immunogenic composition comprising the immunogenic carrier according to any one of [1] to [6], and a short-chain peptide. [8] The immunogenic composition according to [7], wherein the short-chain peptide is chemically bonded to the immunogenic carrier. [9] The immunogenic composition according to [8], wherein the short-chain peptide is chemically bonded to the aminopolysaccharide.

[10] The immunogenic composition according to [8] or [9], wherein the short-chain peptide is chemically bonded to the protein.

[11] The immunogenic composition according to [7] or [8], wherein the short-chain peptide comprises an amino acid sequence derived from a protein selected from the group consisting of a tumor antigen protein, an immune checkpoint protein, and immunoglobulin E.

[12] The immunogenic composition according to

[11] , wherein the short-chain peptide comprises an amino acid sequence derived from PD-L1, an amino acid sequence derived from CTLA-4, or an amino acid sequence derived from immunoglobulin E.

[13] The immunogenic composition according to

[12] , wherein the short-chain peptide is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 5.

[14] The immunogenic composition according to any one of

[11] to

[13] , which is used for treating or preventing a tumor or an atopic disease.

[0007] According to the present invention, there are provided an immunogenic carrier that is highly immunogenic, inexpensive, and highly safe, and an immunogenic composition that uses the immunogenic carrier.

[0008]

[0033] Figure 1 shows the test schedule for Reference Example 1.

[0034] Figure 1 shows the results of measuring the serum anti-mouse antibody concentration of BALB / c mice immunized with KLH-mCTLA4-1 in Reference Example 1.

[0035] Figure 1 shows the results of measuring the serum anti-mouse antibody concentration of BALB / c mice immunized with KLH-mCTLA4-2 in Reference Example 1.

[0036] Figure 1 shows the results of measuring the serum anti-mouse antibody concentration of BALB / c mice immunized with KLH-mCTLA4-1 or KLH-mCTLA4-2 in Reference Example 1.

[0037] Figure 1 shows the results of measuring the tumor size over time after transplantation, after transplantation, with the mouse colon cancer cell line Colon26.

[0038] Figure 1 shows the test schedule for Example 1.

[0039] Figure 1 shows the results of measuring the serum anti-mouse CTLA-4 antibody concentration of BALB / c mice immunized with TT-AD-mCTLA4-1 in Example 1. In Example 2, the results of measuring the anti-mouse CTLA-4 antibody concentration in the serum of BALB / c mice immunized with TT-AD-mCTLA4-1 and the anti-mouse CTLA-4 antibody concentration in the serum of BALB / c mice immunized with KLH-mCTLA4-1 are shown. The test schedule for Example 3 is shown. In Example 3, the results of measuring the anti-human CTLA-4 antibody concentration in the serum of CTLA-4 humanized mice immunized with TT-AD-hCTLA4 are shown. The CTLA4 humanized mice are C57BL / 6 mice into which the human CTLA4 gene has been introduced. The test schedule for Example 4 is shown. In Example 4, the results of measuring the anti-mouse PD-L1 antibody concentration in the serum of C57BL / 6 mice immunized with TT-AD-mPDL1 are shown. In Example 4, the mouse colon cancer cell line MC38 was transplanted into C57BL / 6 mice immunized with TT-AD-mPDL1, and the tumor size after transplantation was measured over time. The test schedule for Example 5 is shown. In Example 5, the results of measuring the serum anti-human PD-L1 antibody concentration in PD-L1-humanized C57BL / 6 mice immunized with TT-AD-hPDL1 are shown. PD-L1-humanized C57BL / 6 mice are C57BL / 6 mice into which the human PD-L1 gene has been incorporated. In Example 5, the mouse colon cancer cell line MC38 was transplanted into PD-L1-humanized C57BL / 6 mice immunized with TT-AD-hPDL1, and the results of measuring the tumor size after transplantation over time are shown.Example 6 shows the results of measuring the serum anti-human CTLA-4 antibody concentration over time after the final boost in CTLA-4 humanized mice immunized with TT-AD-hCTLA4. Example 7 shows the results of measuring the serum anti-human PD-L1 antibody concentration in PD-L1 humanized C57BL / 6 mice immunized with immunogenic compositions prepared using AD400 (400 amino groups / polymer), AD50 (50 amino groups / polymer), or AD25 (25 amino groups / polymer) as the aminopolysaccharide. Example 8 shows the results of measuring the serum anti-human PD-L1 antibody concentration in PD-L1 humanized C57BL / 6 mice immunized with TT-hPDL1 / AD400-hPDL1. 1 shows the results of measuring the serum anti-mouse PDL1 antibody concentration of C57BL / 6 mice immunized with TT-mPDL1 / AD400-mPDL1, TT-mPDL1 / AD50-mPDL1, TT-mPDL1 / AD25-mPDL1, TT-mPDL1 / AD400, or TT-mPDL1 in Example 9. 1 shows the results of measuring the serum anti-mouse IgE antibody concentration of C57BL / 6 mice immunized with TT-mIgE-1 / AD400-mIgE-1 and TT-mIgE-2 / AD400-mIgE-2 in Example 10. 1 is a schematic diagram illustrating the mechanism of induction of antibody production against a target protein by an immunogenic composition of one embodiment. 1 is a schematic diagram illustrating the mechanism of induction of antibody production against a target protein by an immunogenic composition of one embodiment.

[0009] A numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0010] Unless otherwise specified, "a," "an," and "the" are inclusive of singular and plural and are understood to mean "one or more."

[0011] The term "comprise" means that it may contain components other than the target component. The term "consist of" means that it does not contain components other than the target component. The term "consist essentially of" means that it does not contain components other than the target component in a manner that would exert a special function (such as a manner that would completely lose the effects of the invention). In this specification, when "comprise" is used, it includes both "consist of" and "consist essentially of" embodiments.

[0012] Proteins, peptides, nucleic acids, vectors, and cells may be isolated. "Isolated" means in a natural state or separated from other components. "Isolated" may be substantially free of other components. "Substantially free of other components" means that the content of other components contained in the isolated component is negligible. The content of other components contained in the isolated component may be, for example, 10% by mass or less, 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.5% by mass or less, or 0.1% by mass or less. The proteins, peptides, nucleic acids, vectors, and cells described herein may be isolated proteins, isolated peptides, isolated nucleic acids, isolated vectors, and isolated cells, respectively.

[0013] [Immunogenic Carrier] A first aspect of the present disclosure is an immunogenic carrier, which comprises an aminopolysaccharide containing 100 or more amino groups per molecule and a protein.

[0014] An immunogenic carrier refers to a carrier that confers immunogenicity to a hapten. By binding a hapten to an immunogenic carrier, the hapten expresses immunogenicity and induces antibody production when administered to the body. The immunogenic carrier of this embodiment confers immunogenicity to a short-chain peptide of about 3 to 20 amino acids by binding the peptide. The immunogenic carrier may be composed of one type of molecule or a combination of two or more types of molecules.

[0015] <Aminopolysaccharides> Aminopolysaccharides are polysaccharides containing one or more amino groups. The amino groups contained in aminopolysaccharides are preferably primary amino groups. Examples of aminopolysaccharides include aminated polysaccharides in which amino groups have been introduced into polysaccharides, chitosan, chondroitin sulfate, keratan sulfate, heparan sulfate, and dermatan sulfate. Examples of polysaccharides into which amino groups have been introduced in aminated polysaccharides include dextran, glycogen, pullulan, chitin, gellan gum, and hyaluronic acid. The polysaccharide into which amino groups have been introduced may be a polysaccharide derived from a microorganism. Examples of microorganism-derived polysaccharides include bacterial polysaccharides such as capsular polysaccharides of Streptococcus pneumoniae, and viral polysaccharides such as capsular polysaccharides of influenza viruses.

[0016] The aminopolysaccharide used contains 100 or more amino groups (preferably primary amino groups) per aminopolysaccharide molecule. The number of amino groups contained in the aminopolysaccharide is preferably 200 or more, more preferably 300 or more, more preferably 350 or more, and even more preferably 400 or more per aminopolysaccharide molecule. The upper limit of the number of amino groups contained in the aminopolysaccharide is not particularly limited, and examples include 2000 or less, 1000 or less, 800 or less, and 600 or less per aminopolysaccharide molecule. The upper and lower limits can be arbitrarily combined. The number of amino groups contained in the aminopolysaccharide can be, for example, 100 to 2000, 200 to 1000, 300 to 800, and 400 to 600 per aminopolysaccharide molecule. The greater the number of amino groups contained in the aminopolysaccharide, the more short-chain peptides can be bound when preparing an immunogenic composition.

[0017] The molecular weight of the aminopolysaccharide can be large enough to contain 100 or more amino groups per aminopolysaccharide molecule. Examples of the molecular weight of the aminopolysaccharide include 100 kDa or more, preferably 150 kDa or more, more preferably 200 kDa or more, even more preferably 250 kDa or more, and even more preferably 300 kDa or more. The upper limit of the molecular weight of the aminopolysaccharide is not particularly limited, but examples include 2000 kDa or less, 1500 kDa or less, 1000 kDa or less, 800 kDa or less, 700 kDa or less, and 600 kDa or less. The upper and lower limits can be arbitrarily combined. Examples of the molecular weight of the aminopolysaccharide include 100 to 2000 kDa, 200 to 1000 kDa, 200 to 800 kDa, 300 to 700 kDa, and 400 to 600 kDa.

[0018] The aminopolysaccharide is preferably aminodextran. The molecular weight of the aminodextran is not particularly limited, but may be, for example, 100 to 1000 kDa, preferably 200 to 800 kDa, more preferably 300 to 700 kDa, and even more preferably 400 to 600 kDa. A specific example of the molecular weight of aminodextran is 500 kDa. The number of amino groups contained in the aminodextran may be the same as those listed above for the number of amino groups in the aminopolysaccharide.

[0019] The aminopolysaccharides may be used alone or in combination of two or more.

[0020] <Protein (Carrier Protein)> The protein (hereinafter also referred to as "carrier protein") is not particularly limited as long as it can confer immunogenicity to a hapten. Carrier proteins that have conventionally been used as carrier proteins for immunogenic compositions can be used. Examples of carrier proteins include, but are not limited to, bacterial-derived proteins, bacterial toxoids, albumin, keyhole limpet hemocyanin (KLH), and the like. Examples of bacterial-derived proteins and bacterial-derived toxoids include, but are not limited to, diphtheria toxin CRM197, diphtheria toxoid (DT), tetanus toxoid (TT), tetanus toxoid fragment C, pertussis toxoid, Haemophilus influenzae D protein, Escherichia coli heat-labile entrotoxin (LT), Escherichia coli heat-stable enterotoxin (ST), Pseudomonas aeruginosa-derived exotoxin A, Pseudomonas aeruginosa-derived outer membrane complex c (OMPC), Pseudomonas aeruginosa-derived porin, meningococcal transferrin-binding protein, pneumococcal pneumococcal pneumococcal pneumococcal surface protein A (PspA), pneumococcal surface attachment protein A (PsaA), pneumococcal PhtD, pneumococcal BVH-3, and pneumococcal BVH-11. Albumins include, but are not limited to, ovalbumin, human serum albumin, bovine serum albumin (BSA), and the like.

[0021] Among these, bacterial toxoids are preferred as carrier proteins, with tetanus toxoid being more preferred.

[0022] One type of carrier protein may be used alone, or two or more types may be used in combination.

[0023] The aminopolysaccharide and the carrier protein may or may not be chemically bonded. "Chemically bonded" means that the aminopolysaccharide and the carrier protein are bonded directly or indirectly via a covalent bond.

[0024] When the aminopolysaccharide and the carrier protein are chemically bonded, the immunogenic carrier is preferably a bond between the aminopolysaccharide and the carrier protein (a conjugate of the aminopolysaccharide and the carrier protein). When the aminopolysaccharide and the carrier protein are chemically bonded, the aminopolysaccharide and the carrier protein may be bonded directly or indirectly via a linker or the like. For example, the aminopolysaccharide and the carrier protein may be bonded via a linker peptide. As the linker peptide, for example, a peptide of about 10 to 20 amino acids can be used. The linker peptide preferably contains cysteine. By containing cysteine, the thiol group in the cysteine ​​can be used to bond the carrier protein and the linker peptide and / or the aminopolysaccharide and the linker peptide. A specific example of the linker peptide is a peptide containing the amino acid sequence set forth in SEQ ID NO: 1, and a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is preferred. One type of linker peptide may be used alone, or two or more types may be used in combination.

[0025] The aminopolysaccharide and the carrier protein, or the aminopolysaccharide, the carrier protein, and the linker peptide can be bound by known methods, for example, using a crosslinking agent, a condensing agent, or the like.

[0026] Examples of crosslinking agents include those capable of generating active esters, with active esterifying agents being preferred. Examples of crosslinking agents include NHS (N-hydroxysuccinimide), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate)cyclohexane-1-carboxylate), sulfo-NHS (N-hydroxysulfosuccinimide), sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), HoBt (1-hydroxybenzotriazole), 1-hydroxybenzotriazole (HOAt), pentafluorophenol, and the like, but are not limited to these. Among these, crosslinking agents containing an NHS-ester group (sulfo-NHS, sulfo-SMCC, NHS, SMCC, etc.) are preferred.

[0027] The condensing agent may be a carbodiimide-based condensing agent, such as 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), or diisopropylcarbodiimide (DIPC). Of these, EDC is preferred as the condensing agent.

[0028] The aminopolysaccharide and the carrier protein can be conjugated, for example, using a cross-linking agent containing an amine-reactive group (eg, an NHS ester group) and a maleimide group.

[0029] The conjugation of an aminopolysaccharide to a carrier protein can be carried out, for example, using a linker peptide as follows: In the presence of a carbodiimide condensing agent, the carrier protein is reacted with an active esterifying agent to activate the carboxyl groups in the carrier protein. The reaction temperature can be, for example, 10 to 50°C, with 20 to 40°C being preferred. The reaction time can be, for example, 5 to 60 minutes. After the reaction, unreacted carbodiimide condensing agent and active esterifying agent may be removed by gel filtration or the like. Next, the linker peptide is reacted with the active ester introduced into the carrier protein. This allows the carrier protein and the linker peptide to be conjugated. The reaction temperature can be, for example, 10 to 50°C, with 20 to 40°C being preferred. The reaction time can be, for example, 10 to 150 minutes. After the reaction, unreacted linker peptide may be removed by gel filtration or the like. Next, the thiol group of the cysteine ​​residue contained in the conjugate of the carrier protein and the linker peptide is reduced using a reducing agent. Examples of the reducing agent include dithiothreitol and tris(2-carboxyethyl)phosphine (TCEP). The reaction temperature is, for example, 10 to 50°C, preferably 20 to 40°C. The reaction time is, for example, 10 to 150 minutes.

[0030] The aminopolysaccharide is reacted with a crosslinking agent containing an amine-reactive group and a maleimide group to introduce the maleimide group into the aminopolysaccharide. The reaction temperature is, for example, 10 to 50°C, and preferably 20 to 40°C. The reaction time is, for example, 10 to 150 minutes. After the reaction, the crosslinking agent may be removed by gel filtration or the like.

[0031] A conjugate of a reduced carrier protein and a linker peptide is reacted with an aminopolysaccharide to which a maleimide group has been introduced. This allows the carrier protein and the aminopolysaccharide to be bonded. The reaction temperature is, for example, 10 to 50°C, preferably 20 to 40°C. The reaction time is, for example, 10 to 150 minutes.

[0032] When the aminopolysaccharide and the carrier protein are not chemically bonded, the aminopolysaccharide and the carrier protein can be used in combination.

[0033] The mass ratio of the carrier protein to the aminopolysaccharide in the immunogenic carrier is, for example, 1:20 to 20:1, preferably 1:10 to 10:1, and more preferably 1:5 to 5:1. When the aminopolysaccharide and the carrier protein are chemically bonded, the mass ratio of the carrier protein to the aminopolysaccharide is, for example, more preferably 1:3 to 3:1, even more preferably 1:2 to 2:1, and even more preferably 1:1. When the aminopolysaccharide and the carrier protein are not chemically bonded, the mass ratio of the carrier protein to the aminopolysaccharide is, for example, preferably 2:1 to 5:1.

[0034] The immunogenic carrier of this embodiment can impart strong immunogenicity to substances with low immunogenicity, such as haptens and short-chain peptides of approximately 3 to 20 amino acids. Furthermore, because it is composed of proteins and polysaccharides, it is highly safe and can be produced inexpensively.

[0035] [Immunogenic composition] A second aspect of the present disclosure is an immunogenic composition, which comprises the immunogenic carrier according to the first aspect and a short-chain peptide.

[0036] <Immunogenic Carrier> The immunogenic carrier is the immunogenic carrier according to the first aspect. The explanation of the immunogenic carrier is the same as above. One type of immunogenic carrier may be used alone, or two or more types may be used in combination.

[0037] <Short-chain peptide> In this specification, "short-chain peptide" refers to a peptide of 30 amino acids or less, preferably a peptide of about 3 to 25 amino acids. Short-chain peptides can be easily produced by known peptide synthesis methods. Short-chain peptides are less likely to cause unexpected immune reactions and are highly safe. Short-chain peptides are preferably about 5 to 20 amino acids, more preferably about 10 to 20 amino acids.

[0038] The short peptide can be a peptide derived from a target protein that is the target of the immune response induced by the immunogenic composition. The target protein can be selected appropriately depending on the purpose. A "peptide derived from a target protein" means a peptide that contains a portion of the amino acid sequence of the target protein. A peptide derived from a target protein may contain only a portion of the amino acid sequence of the target protein, or may contain other sequences in addition to the amino acid sequence of the target protein. The portion of the amino acid sequence of the protein contained in a peptide derived from a target protein can be 3 to 20 amino acids, and preferably 5 to 12 amino acids.

[0039] For the purpose of treating non-infectious diseases, the target protein from which the short peptide is derived is preferably a protein derived from an organism of the same species as the target organism. When the target organism of the immunogenic composition of this embodiment is a human, the target protein is preferably derived from a human. Examples of target proteins include proteins involved in diseases. Examples of target proteins include proteins whose activity is inhibited to alleviate or improve symptoms caused by diseases.

[0040] Examples of short-chain peptides include peptides comprising an amino acid sequence derived from a protein selected from the group consisting of a tumor antigen protein and an immune checkpoint protein. Tumor immunity can be induced by using a peptide comprising an amino acid sequence derived from a tumor antigen protein. Antibodies against immune checkpoint proteins can be produced by using a peptide comprising an amino acid sequence derived from an immune checkpoint protein, allowing the antibodies to function as immune checkpoint inhibitors.

[0041] Preferably, the tumor antigen protein is highly expressed in tumor cells and is not or hardly expressed in normal cells. Examples of tumor antigen proteins include, but are not limited to, CD20, EGFR, CD19, CD22, CD33, PSMA, EGFR variant, HER2, CEA, mesothelin, CD7, CD10, CD30, CD34, CD38, CD41, CD44, CD74, CD123, CD133, CD171, CS1 (CD319), IL-13Ra2, BCMA, PSCA, EpCAM, and GPC3.

[0042] Immune checkpoint proteins are proteins that have the function of suppressing immune responses. Examples of immune checkpoint proteins include, but are not limited to, PD-L1, PD-L2, PD-1, CTLA-4, TIM-3, Galectin-9, BTLA, LAG-3, and Siglec-10.

[0043] The short-chain peptide may be, for example, a peptide containing an amino acid sequence derived from immunoglobulin E (IgE). By using a peptide containing an amino acid sequence derived from IgE, antibodies against IgE can be produced, thereby alleviating or ameliorating the symptoms of atopic diseases, such as bronchial asthma, seasonal allergic rhinitis, sudden chronic urticaria, and atopic dermatitis.

[0044] The short-chain peptide may be a peptide containing an amino acid sequence derived from tumor necrosis factor (TNF)-α. By using a peptide containing an amino acid sequence derived from TNF-α, antibodies against TNF-α can be produced, thereby alleviating or ameliorating the symptoms of inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, ulcerative colitis, and ankylosing spondylitis.

[0045] The short peptide may be a peptide containing an amino acid sequence derived from a protein of a pathogenic bacterium or a pathogenic virus. By using a peptide containing an amino acid sequence derived from a protein of a pathogenic bacterium or a pathogenic virus, antibodies against the pathogenic bacterium or virus can be produced, thereby suppressing the growth of the pathogenic bacterium or virus.

[0046] The short-chain peptide is preferably chemically bound to at least one of the immunogenic carriers, an aminopolysaccharide and a carrier protein. "Chemically bound" means that the aminopolysaccharide or the carrier protein and the short-chain peptide are directly or indirectly bound by a covalent bond. For this binding, the short-chain peptide preferably has a cysteine ​​residue, more preferably at either or both of the N-terminus and the C-terminus. The presence of a cysteine ​​residue facilitates binding with a crosslinker containing a maleimide group.

[0047] When the amino acid sequence derived from the target protein does not have a cysteine ​​residue, one to several (for example, about 1 to 10, preferably about 1 to 5, more preferably about 1 to 3) amino acid residues including a cysteine ​​residue may be added to either or both of the N-terminus and the C-terminus of the amino acid sequence derived from the target protein.

[0048] The short-chain peptide is preferably a peptide comprising an amino acid sequence derived from an immune checkpoint protein, and is preferably a peptide derived from PD-L1 (programmed cell death ligand 1) or CTLA-4 (cytotoxic T-lymphocyte associated protein 4). Examples of peptides comprising an amino acid sequence derived from human PD-L1 include peptides comprising the amino acid sequence set forth in SEQ ID NO: 3. The amino acid sequence set forth in SEQ ID NO: 3 is an example of an amino acid sequence derived from human PD-L1. A peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 may have approximately 1 to 10 amino acid residues (preferably 1 to 5 amino acids, more preferably 1 to 3 amino acids) added to either or both of the N-terminus and C-terminus. The added amino acid residues are not particularly limited, but preferably contain a cysteine ​​residue. For example, amino acid residues may be added so that a cysteine ​​residue is located at either or both of the N-terminus and C-terminus. Such peptides include, for example, peptides comprising the amino acid sequence set forth in SEQ ID NO: 2, with a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 being preferred. Peptides comprising an amino acid sequence derived from human CTLA-4 include peptides comprising the amino acid sequence set forth in SEQ ID NO: 5. The amino acid sequence set forth in SEQ ID NO: 5 is an example of an amino acid sequence derived from human CTLA-4. A peptide comprising the amino acid sequence set forth in SEQ ID NO: 5 may have approximately 1 to 10 amino acid residues (preferably 1 to 5 amino acids, more preferably 1 to 3 amino acids) added to either or both of the N-terminus and C-terminus. The added amino acid residues are not particularly limited, but preferably contain cysteine ​​residues. For example, amino acid residues may be added so that cysteine ​​residues are located at either or both of the N-terminus and C-terminus. Such peptides include, for example, peptides comprising the amino acid sequence set forth in SEQ ID NO: 4, with a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 being preferred.

[0049] The short-chain peptides may be used alone or in combination of two or more.

[0050] The short-chain peptide is preferably chemically bound to the immunogenic carrier, and is preferably a conjugate of the immunogenic carrier and the short-chain peptide (hereinafter also referred to as an "immunogenic conjugate"). The short-chain peptide can be bound to the immunogenic carrier by a known method. The binding can be carried out using, for example, a crosslinking agent, a condensing agent, etc. Examples of crosslinking agents and condensing agents include those similar to those described above.

[0051] Immunogenic carriers and short-chain peptides can be bound, for example, as follows: A crosslinker containing an amine-reactive group and a maleimide group is reacted with the immunogenic carrier to introduce a maleimide group into the immunogenic carrier. The reaction temperature can be, for example, 10 to 50°C, with 20 to 40°C being preferred. The reaction time can be, for example, 10 to 150 minutes. After the reaction, the crosslinker can be removed by gel filtration or the like. Examples of crosslinkers containing an amine-reactive group and a maleimide group include crosslinkers containing an NHS-ester group (sulfo-NHS, sulfo-SMCC, NHS, SMCC, etc.).

[0052] The thiol group of the cysteine ​​residue contained in the short-chain peptide is reduced using a reducing agent. Examples of reducing agents include dithiothreitol and tris(2-carboxyethyl)phosphine (TCEP). The reaction temperature is, for example, 10 to 50°C, preferably 20 to 40°C. The reaction time is, for example, 10 to 150 minutes.

[0053] A reduced short-chain peptide is reacted with an immunogenic carrier into which a maleimide group has been introduced. This allows the short-chain peptide to be bound to the immunogenic carrier. The reaction temperature is, for example, 10 to 50°C, preferably 20 to 40°C. The reaction time is, for example, 10 to 150 minutes. After the reaction, unreacted short-chain peptide can be removed by dialysis or the like.

[0054] When an immunogenic carrier is used in which the carrier protein and the aminopolysaccharide are not chemically bound, the binding of the carrier protein to the short-chain peptide and the binding of the aminopolysaccharide to the short-chain peptide may be carried out separately. After the binding reaction with the short-chain peptide, the carrier protein bound to the short-chain peptide and the aminopolysaccharide bound to the short-chain peptide can be mixed to prepare an immunogenic composition.

[0055] When an immunogenic carrier in which a carrier protein and an aminopolysaccharide are chemically bonded is used, the mass ratio of the immunogenic carrier to the short-chain peptide is, for example, 1:1 to 100:1, preferably 5:1 to 50:1, more preferably 15:1 to 30:1, and even more preferably 20:1. The mass ratio of the carrier protein to the short-chain peptide in the immunogenic carrier is, for example, 1:1 to 50:1, preferably 3:1 to 20:1, more preferably 5:1 to 15:1, even more preferably 8:1 to 12:1, and particularly preferably 10:1. The mass ratio of the aminopolysaccharide to the short-chain peptide in the immunogenic carrier is, for example, 1:1 to 50:1, preferably 3:1 to 20:1, more preferably 5:1 to 15:1, even more preferably 8:1 to 12:1, and particularly preferably 10:1.

[0056] When an immunogenic carrier in which the carrier protein and aminopolysaccharide are not chemically bonded is used, the mass ratio of the carrier protein to the short-chain peptide is, for example, 1:1 to 50:1, preferably 3:1 to 20:1, more preferably 5:1 to 15:1, even more preferably 8:1 to 12:1, and particularly preferably 10:1. The mass ratio of the aminopolysaccharide to the short-chain peptide is, for example, 1:1 to 50:1, preferably 3:1 to 20:1, more preferably 5:1 to 15:1, and even more preferably 1:1 to 5:1. When a conjugate of a carrier protein and a short-chain peptide and a conjugate of an aminopolysaccharide and a short-chain peptide are mixed, the mass ratio is, for example, 1:20 to 20:1, preferably 1:10 to 10:1, more preferably 1:5 to 5:1, even more preferably 1:1 to 5:1, and particularly preferably 2:1 to 5:1.

[0057] <Optional Components> The immunogenic composition of this embodiment may contain optional components in addition to the above components. Examples of optional components include pharmaceutically acceptable carriers. A "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of the active ingredient and is not substantially toxic to the recipient. "Not substantially toxic" means that the ingredient is not toxic to the recipient at a dose normally used. In the immunogenic composition of this embodiment, a pharmaceutically acceptable carrier is a carrier that does not inhibit the function of the immunogenic carrier and the short-chain peptide (a conjugate of the immunogenic carrier and the short-chain peptide) and is not substantially toxic to the recipient. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable components that are typically considered to be inactive ingredients. Pharmaceutically acceptable carriers include, but are not limited to, solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, and fillers. One pharmaceutically acceptable carrier may be used alone, or two or more may be used in combination. The immunogenic composition of this embodiment may contain, for example, a buffer solution as a solvent. Examples of buffer solutions include phosphate buffer (PB), phosphate-buffered saline (PBS), acetate buffer, citrate buffer, citrate-phosphate buffer, borate buffer, tartrate buffer, Tris buffer, and HEPES buffer.

[0058] The immunogenic composition may contain other components in addition to the above components. The other components are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Examples of other components include pharmaceutical additives other than those mentioned above. Examples of pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. These components may be used alone or in combination of two or more.

[0059] The immunogenic composition may contain any active ingredient. Examples of active ingredients include, but are not limited to, antiviral agents, antibiotics, anti-inflammatory agents, antipyretics, analgesics, etc. One active ingredient may be used alone, or two or more active ingredients may be used in combination.

[0060] The immunogenic composition may contain an adjuvant. Examples of adjuvants include, but are not limited to, aluminum hydroxide, nucleic acid, monophosphoryl lipid A, chitosan, saponin, etc. One type of adjuvant may be used alone, or two or more types may be used in combination.

[0061] The dosage form of the immunogenic composition is not particularly limited and can be any dosage form commonly used as a pharmaceutical preparation. The immunogenic composition of this embodiment may be an oral formulation or a parenteral formulation, with parenteral formulations being preferred. Oral formulations include, for example, tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, emulsions, etc. Parenteral formulations include, for example, injections, drip infusions, suppositories, nasal drops, enteral preparations, inhalants, etc. Immunogenic compositions in these dosage forms can be formulated according to standard methods (e.g., methods described in the Japanese Pharmacopoeia).

[0062] The route of administration of the immunogenic composition of this embodiment is not particularly limited and may be oral or parenteral, with parenteral administration being preferred. Examples of parenteral administration include sublingual administration, intravenous administration, intranasal administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, and enteral administration.

[0063] The immunogenic composition can be administered in a therapeutically effective amount of the immunogenic carrier and short peptide. The term "therapeutically effective amount" refers to an amount of drug effective for treating or preventing a target disease. For example, a therapeutically effective amount of the immunogenic carrier and short peptide (immunogenic conjugate) can be an amount effective for inducing antibody production against a target protein. The therapeutically effective amount can be determined appropriately based on the patient's symptoms, weight, age, and sex, as well as the dosage form and administration method of the pharmaceutical composition. For example, the immunogenic composition can be administered in a single dose of 0.0001 to 1000 μg of the immunogenic carrier and short peptide (immunogenic conjugate) per kg of the subject's body weight. The dose may be 0.001 to 800 μg / kg, 0.05 to 500 μg / kg, 0.05 to 100 μg / kg, 0.1 to 50 μg / kg, or 0.1 to 10 μg / kg. The single dose of the immunogenic conjugate for an adult human is, for example, 0.1 to 1000 μg, or may be 1 to 500 μg, 1 to 200 μg, 10 to 100 μg, or 2 to 70 μg.

[0064] The immunogenic composition may contain a therapeutically effective amount of an immunogenic carrier and a short-chain peptide (immunogenic conjugate) per unit dosage form. For example, the content of the immunogenic carrier and the short-chain peptide (immunogenic conjugate) in the immunogenic composition may be 0.01 to 90% by mass, 0.05 to 80% by mass, or 0.1 to 60% by mass.

[0065] The immunogenic composition may be administered in a single dose or multiple doses. In the case of multiple doses, the administration interval may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., as well as the dosage form and administration method of the immunogenic composition, etc. The administration interval may be, for example, once a week, once a month, once every few months, etc.

[0066] The subject to which the immunogenic composition is administered is not particularly limited. The subject to which the immunogenic composition is administered is preferably a mammal, and may be a human or a mammal other than a human. Examples of mammals other than a human include primates (monkeys, gorillas, chimpanzees, marmosets, etc.), rodents (mice, rats, guinea pigs, hamsters, etc.), pets (dogs, cats, rabbits, ferrets, etc.), and livestock (cows, pigs, horses, goats, sheep, etc.).

[0067] The disease to which the immunogenic composition is applied is selected depending on the type of short peptide. The immunogenic composition is preferably administered to a disease associated with the target protein from which the short peptide is derived. When the short peptide is derived from a protein selected from the group consisting of tumor antigen proteins and immune checkpoint proteins, the immunogenic composition can be used to treat or prevent tumors. Examples of tumors include epithelial tumors such as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, and ovarian cancer; osteosarcomas such as chondrosarcoma and Ewing's sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma; and blood cancers such as malignant lymphoma, leukemia, and multiple myeloma.

[0068] When the short-chain peptide is a peptide derived from immunoglobulin E, the immunogenic composition can be used to treat or prevent atopic diseases, such as bronchial asthma, seasonal allergic rhinitis, sudden chronic urticaria, and atopic dermatitis.

[0069] The immunogenic composition of this embodiment uses the immunogenic carrier according to the first aspect, thereby imparting high immunogenicity to short peptides. Therefore, the immunogenic composition is highly effective in inducing antibody production against the target protein from which the short peptide is derived in vivo. Furthermore, antibody production quickly ceases when administration of the immunogenic composition of this embodiment is stopped. Therefore, the immunogenic composition of this embodiment can be said to be highly safe.

[0070] 20 and 21 are schematic diagrams illustrating the mechanism by which the immunogenic composition of this embodiment induces antibody production against a target protein.

[0071] FIG. 20 illustrates an immunogenic composition 10 comprising an immunogenic carrier in which an aminopolysaccharide AS and a carrier protein CP are chemically bonded. In the immunogenic composition 10, a short peptide SP is chemically bonded to both the aminopolysaccharide AS and the carrier protein CP. The short peptide SP comprises a peptide derived from a target protein TP. In a subject administered with the immunogenic composition 10, the immunogenic composition 10 is taken up by dendritic cells (DC). The carrier protein CP is fragmented intracellularly within the dendritic cells DC to form carrier protein-derived peptides (CPPs). The dendritic cells DC present the carrier protein-derived peptides (CPPs) via MHC class II molecules. Helper T cells (Th) having a T cell receptor (TCR) capable of recognizing the carrier protein-derived peptides (CPPs) recognize and become activated by the carrier protein-derived peptides (CPPs) presented by the DCs.

[0072] Meanwhile, B cells B1 having a B cell receptor (BCR1) capable of binding to the short peptide SP recognize the short peptide SP contained in the immunogenic composition 10 via BCR1 and take up the immunogenic composition 10. The carrier protein CP is fragmented intracellularly within the B cell B1 to form the carrier protein-derived peptide CPP. The B cell B1 presents the carrier protein-derived peptide CPP via an MHC class II molecule. Helper T cells Th having a T cell receptor (TCR) capable of recognizing the carrier protein-derived peptide CPP, which are activated by antigen presentation by dendritic cells DC, recognize the carrier protein-derived peptide CPP presented on the B cell B1 and activate the B cell B1. This induces the production of an antibody Ab1 against the target protein TP by the B cell B1. Similarly, the production of an antibody Ab2 against the carrier protein CP is also induced by B cells B2 having a B cell receptor (BCR2) capable of binding to the carrier protein CP. The immunogenic composition 10 contains an aminopolysaccharide AS bound to a short peptide SP, which promotes the activation of dendritic cells DC that have taken up the immunogenic composition 10. The activation of B cells B1 that have taken up the immunogenic composition 10 is also promoted. Furthermore, the binding of the short peptide SP bound to the aminopolysaccharide AS to BCR1 promotes the activation of B cells B1. Furthermore, the disappearance of the immunogenic composition 10 from the body makes it impossible to maintain helper T cells Th that can recognize the carrier protein-derived peptide CPP, thereby halting antibody production by B cells B1.

[0073] FIG. 21 shows an immunogenic composition 20 containing an immunogenic carrier in which the aminopolysaccharide AS and the carrier protein CP are not chemically bound. In the immunogenic composition 20, the short peptide SP is chemically bound to both the aminopolysaccharide AS and the carrier protein CP. The short peptide SP contains a peptide derived from the target protein TP. In a subject administered with the immunogenic composition 20, the immunogenic composition 20 is taken up by dendritic cells (DC). The carrier protein CP is fragmented intracellularly in the dendritic cells DC to form carrier protein-derived peptides (CPPs). The dendritic cells DC present the carrier protein-derived peptides (CPPs) using MHC class II molecules. Helper T cells (Th) with T cell receptors (TCRs) capable of recognizing the carrier protein-derived peptides (CPPs) recognize and become activated by the carrier protein-derived peptides (CPPs) presented by the DCs.

[0074] Meanwhile, B cells B1 having a B cell receptor (BCR1) capable of binding to the short peptide SP recognize the short peptide SP via BCR1 and take up the aminopolysaccharide AS to which the short peptide SP is bound and the carrier protein CP to which the short peptide is bound. The carrier protein CP is fragmented within the B cell B1 to form the carrier protein-derived peptide CPP. The B cell B1 presents the carrier protein-derived peptide CPP using MHC class II molecules. Helper T cells Th having a T cell receptor (TCR) capable of recognizing the carrier protein-derived peptide CPP, activated by antigen presentation by dendritic cells DC, recognize the carrier protein-derived peptide CPP presented on the B cell B1 and activate the B cell B1. This induces the production of antibody Ab1 against the target protein TP by the B cell B1. Similarly, the production of antibody Ab2 against the carrier protein CP is also induced by B cells B2 having a B cell receptor (BCR2) capable of binding to the carrier protein CP.

[0075] When the immunogenic composition 20 contains an aminopolysaccharide AS bound to a short peptide SP, in addition to the carrier protein CP bound to the short peptide SP, the aminopolysaccharide AS bound to the short peptide SP is also taken up by dendritic cells DC, promoting the activation of dendritic cells DC. Furthermore, the binding of the short peptide SP bound to the aminopolysaccharide AS to the BCR1 of B cells B1 promotes the activation of B cells B1. Furthermore, the binding of the short peptide SP bound to the aminopolysaccharide AS to BCR1 and its uptake by B cells B1 promotes the activation of B cells B1. Furthermore, the disappearance of the immunogenic composition 10 from the body prevents the maintenance of helper T cells Th capable of recognizing the carrier protein-derived peptide CPP, thereby halting antibody production by B cells B1.

[0076] As described above, the immunogenic composition of this embodiment can promote antibody production against a target protein by comprising an immunogenic carrier including an aminopolysaccharide and a carrier protein, and a short-chain peptide bound to the immunogenic carrier.

[0077] [Other Aspects] In one aspect, the present disclosure provides a method for treating or preventing a tumor, comprising administering to a subject an effective amount of a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 5. In one aspect, the present disclosure provides use of a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 5 in the manufacture of a pharmaceutical composition for treating or preventing a tumor. In one aspect, the present disclosure provides a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 5 for use in treating or preventing a tumor. In one aspect, the present disclosure provides use of a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 5 for treating or preventing a tumor. In one aspect, the present disclosure provides an anti-cancer agent containing a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 5. In one aspect, the present disclosure provides an agent for inducing anti-tumor immunity containing a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 5. The effective amount may be a therapeutically effective amount of the peptide. The subject is a subject in need of tumor treatment or prevention. The peptide may be a conjugate with an immunogenic carrier according to the first aspect. The peptide may be a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4.

[0078] In one aspect, the present disclosure provides a method for inducing antibody production against a short-chain peptide, comprising administering to a subject an immunogenic composition comprising an immunogenic carrier comprising an aminopolysaccharide containing 100 or more amino groups per molecule and a protein, and the short-chain peptide. In one aspect, the present disclosure provides a method for inducing anti-tumor immunity, comprising administering to a subject an immunogenic composition comprising an immunogenic carrier comprising an aminopolysaccharide containing 100 or more amino groups per molecule and a protein, and a short-chain peptide, wherein the short-chain peptide comprises a peptide derived from a protein selected from the group consisting of a tumor antigen protein and an immune checkpoint protein.

[0079] In one aspect, the present disclosure provides a method for treating or preventing a disease in which a target protein comprising a partial peptide, said short-chain peptide, is involved in the pathology, comprising administering to a subject an immunogenic composition comprising an immunogenic carrier comprising an aminopolysaccharide comprising 100 or more amino groups per molecule and a protein, and a short-chain peptide. In one aspect, the present disclosure provides a method for treating or preventing a tumor, comprising administering to a subject an immunogenic composition comprising an immunogenic carrier comprising an aminopolysaccharide comprising 100 or more amino groups per molecule and a protein, and a short-chain peptide, wherein the short-chain peptide comprises a peptide derived from a protein selected from the group consisting of tumor antigen proteins and immune checkpoint proteins. In one aspect, the present disclosure provides a method for treating or preventing an atopic disease, comprising administering to a subject an immunogenic composition comprising an immunogenic carrier comprising an aminopolysaccharide comprising 100 or more amino groups per molecule and a protein, and a short-chain peptide, wherein the short-chain peptide comprises a peptide derived from immunoglobulin E.

[0080] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0081] <Preparation of Immunogenic Composition (1)> <Short-chain peptides: human PD-L1, mouse PD-L1> (Materials) Tetanus toxoid (TT) 500 kDa Aminodextran (AD) (400 amino groups / polymer) (AD500x400, Fina Biosolutions) Linker peptide ([H]KGSGSGSGSC[OH]; SEQ ID NO: 1) Human PD-L1 peptide (hPDL1_V) ([H]CSGLIVYWEMEDKNGSC[OH]; SEQ ID NO: 2), human PD-L1-derived portion: LIVYWEMEDKN (SEQ ID NO: 3) Mouse PD-L1 peptide (mPDL1_V) ([H]CSGLVVYWEKEDEQGSC[OH]; SEQ ID NO: 6), mouse PD-L1-derived portion: LVVYWEKEDEQ (SEQ ID NO: 7) [H] is the N-terminus of the peptide (-NH ２ ), and [OH] represents the C-terminus of the peptide (—COOH). The above materials were prepared in a conjugation buffer (50 mM Na ２ HPO４ , 50 mM NaH ２ P.O. ４ , 150 mM NaCl, 10 mM EDTA, pH 7.4).

[0082] (Conjugation of linker peptide to tetanus toxoid) Tetanus toxoid (1 mg) was mixed with 2.2 mg of sulfo-NHS (Thermo Fisher Scientific) and 0.8 mg of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) (Tokyo Chemical Industry) in 0.67 mL of conjugation buffer and incubated at room temperature for 15 minutes to activate the carboxyl groups in the tetanus toxoid. Free sulfo-NHS and EDC were removed by gel filtration using a Sephadex G-25 desalting column. The resulting tetanus toxoid-containing solution was mixed with 0.1 mg of linker peptide dissolved in 0.1 mL of conjugation buffer and incubated at room temperature for 1.5 hours. This resulted in a conjugate of the linker peptide and tetanus toxoid (TT-linker).

[0083] Conjugation of aminodextran to TT-linker: A 1 M aqueous solution of dithiothreitol (DTT) (Nacalai Tesque) was prepared using MilliQ water. 0.77 mL of the conjugation buffer containing the TT-linker was mixed with 6 μL of 1 M DTT aqueous solution and incubated at room temperature for 1.5 hours to reduce the cysteine ​​in the TT-linker. 1 mg of aminodextran dissolved in 1 mL of the conjugation buffer was mixed with 0.5 mg of sulfo-SMCC (Thermo Fisher Scientific) dissolved in 100 μL of MilliQ water and incubated at room temperature for 1.5 hours to introduce maleimide groups to the amino groups in the aminodextran. Free DTT and sulfo-SMCC were removed by gel filtration using a Sephadex G-25 desalting column. The resulting solution containing maleimide-activated aminodextran was mixed with 0.77 mL of reduced TT-linker and incubated at room temperature for 1.5 h to generate a conjugate of TT-linker and aminodextran (TT-linker-AD).

[0084] (Conjugation of short peptides to TT-linker-AD) Conjugation buffer (1.7 mL) containing TT-linker-AD was mixed with 100 μL of MilliQ water containing 0.5 mg of sulfo-SMCC and incubated at room temperature for 1.5 hours to introduce maleimide groups to the amino groups in TT-linker-AD. Free sulfo-SMCC was removed by gel filtration using a Sephadex G-25 desalting column. Conjugation buffer (0.1 mL) containing short peptides (0.1 mg) (hPDL1_V or mPDL1_V) was added to 400 μL of TCEP gel (Pierce ＴＭThe resulting mixture was mixed with Immobilized TCEP Disulfide Reducing Gel (Thermo Fisher Scientific) and incubated at room temperature for 1.5 hours with stirring to reduce the terminal cysteines of the short peptides. The mixture was then centrifuged at 500 × g for 2 minutes. The supernatant containing the reduced peptide was mixed with 1.7 mL of conjugation buffer containing maleimide-activated TT-linker-AD and incubated at room temperature for 1.5 hours to conjugate the short peptides to the TT-linker-AD. This yielded a conjugate of TT-linker-AD and the short peptide (TT-linker-AD-peptide). The weight ratio of tetanus toxoid:linker peptide:aminodextran:short peptide was 1:0.1:1:0.1. Unconjugated short peptides were removed by dialysis.

[0085] The immunogenic compositions prepared using the human PD-L1 peptide (hPDL1_V) and mouse PD-L1 peptide (mPDL1_V) as short peptides are described as follows: hPDL1_V: TT-AD-hPDL1 mPDL1_V: TT-AD-mPDL1

[0086] <Preparation of Immunogenic Composition (2)> <Short-chain peptides: human CTLA-4, mouse CTLA-4> (Materials) Tetanus toxoid (TT) 500 kDa Aminodextran (AD) (400 amino groups / polymer) (AD500x400, Fina Biosolutions) Human CTLA-4 peptide (hCTLA4_V) ([H]CSGNELTFLDDSLC[OH]; SEQ ID NO: 4), human CTLA4-derived portion: NELTFLDDSLC (SEQ ID NO: 5) Mouse CTLA-4 peptide 1 (mCTLA4-1_V) ([H]CEYSPSHNTDEVRVSGC[OH]; SEQ ID NO: 8), mouse CTLA4-derived portion: CEYSPSHNTDEVRV (SEQ ID NO: 9) Mouse CTLA-4 peptide 2 (mCTLA4-2_V) ([H]CTTFTEKNTVGSC[OH]; SEQ ID NO: 10), mouse CTLA4-derived portion: TTFTEKNTVG (SEQ ID NO: 11) The above materials were prepared in a conjugation buffer (50 mM Na ２HPO ４ , 50 mM NaH ２ P.O. ４ , 150 mM NaCl, 10 mM EDTA, pH 7.4).

[0087] (Conjugation of aminodextran to tetanus toxoid) A conjugate of aminodextran and tetanus toxoid (TT-AD) was produced in the same manner as described above (Conjugation of aminodextran to TT-linker), except that tetanus toxoid was used instead of the TT-linker.

[0088] (Conjugation of short-chain peptides to TT-AD) Conjugates of TT-AD and peptides were obtained in the same manner as described above (Conjugation of peptides to TT-linker-AD), except that TT-AD was used instead of TT-linker-AD and hCTLA4_V, mCTLA4-1_V, or mCTLA4-2_V was used as the peptide.

[0089] Immunogenic compositions prepared using short peptides, human CTLA-4 peptide (hCTLA4_V), mouse CTLA-4 peptide 1 (mCTLA4-1_V), and mouse CTLA-4 peptide 2 (mCTLA4-2_V), are described as follows: hCTLA4_V: TT-AD-hCTLA4 mCTLA4-1_V: TT-AD-mCTLA4-1 mCTLA4-2_V: TT-AD-mCTLA4-2

[0090] Immunization: Mice were subcutaneously immunized with the immunogenic composition (10-20 μg) dissolved in 100 μL of PBS together with 100 μg of quillaja bark saponin (Nacalai). Two weeks after the primary immunization, the mice were boosted with the same immunogenic composition. Boosting was performed every two weeks, a total of two or three times.

[0091] <ELISA> The amount of antibodies induced by the immunogenic composition in the serum obtained from immunized mice was measured by ELISA. The protein from which the short peptide was derived (recombinant human PD-L1 protein, recombinant human CTLA4 protein, recombinant mouse PD-L1 protein, or recombinant mouse CTLA4 protein (all from COSMO BIO CO., LTD.)) was dissolved in PBS to a concentration of 2 μg / mL. This was used to coat an ELISA plate (Thermo Fisher Scientific). The ELISA plate was then blocked with PBS containing 1% BSA. After washing the wells, serially diluted mouse serum was added to the wells of the ELISA plate and incubated. After washing the wells, alkaline phosphatase-labeled goat anti-mouse IgG (SBA) was added to the wells of the ELISA plate and incubated. After washing the wells, alkaline phosphatase substrate (Sigma-Aldrich) solution was added to the wells and incubated, and the absorbance at 405 nm was measured using a microplate reader (Molecular Devices).

[0092] <Tumor cell transplantation> Colon26 (10 ７ cells) or the mouse colon cancer cell line MC38 (10 ７ The tumor volumes were measured every two days using a digital caliper and expressed as [(width) / (width of the tumor cells)]. ２ × length) / 2.

[0093] Reference Example 1 Evaluation of the Antitumor Effect of Immunogenic Compositions Using Mouse CTLA-4 Peptides To identify mouse CTLA-4 peptides that exhibit antitumor effects, immunogenic compositions were prepared using multiple types of mouse CTLA-4 peptides with a length of 10 to 15 amino acids and keyhole limpet hemocyanin (KLH). BALB / c mice were immunized with these immunogenic compositions, and then the mouse colon cancer cell line Colon26 (10 ７After transplantation, the size of the tumor in the BALB / c mice was measured over time. The test schedule is shown in Figure 1.

[0094] Of the mouse CTLA-4 peptides used in the study, a peptide (mCTLA4-1) consisting of the amino acid sequence set forth in SEQ ID NO: 9 and a peptide (mCTLA4-2) consisting of the amino acid sequence set forth in SEQ ID NO: 11 were identified as short peptides exhibiting antitumor effects. Amino acids such as cysteine ​​were added to mCTLA4-1 to form a peptide (mCTLA4-1_V) consisting of the amino acid sequence set forth in SEQ ID NO: 8. An immunogenic composition containing mCTLA4-1_V (KLH-mCTLA4-1) was prepared using KLH as a carrier. An amino acid such as cysteine ​​was added to mCTLA4-2 to form a peptide (mCTLA4-2_V) consisting of the amino acid sequence set forth in SEQ ID NO: 11. An immunogenic composition containing mCTLA4-2_V (KLH-mCTLA4-2) was prepared using KLH as a carrier.

[0095] Figures 2A and 2B show serum anti-mouse CTLA-4 antibody concentrations in mice immunized with KLH-mCTLA4-1 (Figure 2A) or KLH-mCTLA4-2 (Figure 2B). It was confirmed that serum anti-mouse CTLA-4 antibody concentrations increased one week after the second boost. Figure 3 shows the time course of tumor size in mice immunized with KLH-mCTLA4-1 or KLH-mCTLA4-2. Tumor growth was suppressed in both mice immunized with KLH-mCTLA4-1 and mice immunized with KLH-mCTLA4-2 compared to mice immunized with the control peptide. These results confirmed that KLH-mCTLA4-1 and KLH-mCTLA4-2 have antitumor effects.

[0096] Example 1 (Evaluation of anti-CTLA-4 antibody production by immunogenic composition using mouse CTLA-4 peptide) An immunogenic composition (TT-AD-mCTLA4-1) was prepared using mCTLA4-1 (SEQ ID NO: 8) by the method described above in <Preparation of immunogenic composition (2)>. BALB / c mice were immunized with this immunogenic composition. The test schedule is shown in Figure 4.

[0097] Figure 5 shows the serum anti-mouse CTLA-4 antibody concentration in mice immunized with TT-AD-mCTLA4-1. It was confirmed that the serum anti-mouse CTLA-4 antibody concentration increased one week after the first boost. These results confirmed that the use of TT-AD as an immunogenic carrier can induce the production of anti-CTLA-4 antibodies more quickly than KLH. Furthermore, since the anti-tumor effect of mCTLA4-1 was confirmed by an immunogenic composition using KLH (KLH-mCTLA4-1), it was speculated that TT-AD-mCTLA4-1, which has a high anti-CTLA-4 antibody production-inducing effect, would also have an anti-tumor effect.

[0098] Example 2 Evaluation of the immunostimulatory activity of TT-AD Mice were immunized with TT-AD-mCTLA4-1 and KLH-mCTLA4-1, and the effects of inducing anti-CTLA4 antibody production were compared. Serum was collected from the immunized mice, and the concentration of anti-mouse CTLA-4 antibody in the serum was measured by ELISA.

[0099] The results are shown in Figure 6. In mice immunized with TT-AD-mCTLA4-1, it was confirmed that serum anti-mouse CTLA4 antibody concentrations increased one week after the first boost. On the other hand, in the case of KLH-mCTLA4-1, serum anti-mouse CTLA4 antibody concentrations remained low one week after the first boost. These results confirmed that the use of TT-AD as an immunogenic carrier enables rapid induction of immunity after inoculation of the immunogenic composition.

[0100] Example 3 (Evaluation of anti-CTLA-4 antibody production by immunogenic compositions using human CTLA-4 peptides) In order to identify human CTLA-4 peptides that exhibit the effect of inducing anti-CTLA-4 antibody production, immunogenic compositions were prepared using several types of human CTLA-4 peptides with a length of 10 to 15 amino acids by the method described above in <Preparation of immunogenic compositions (2)>. CTLA-4-humanized mice were immunized with these immunogenic compositions. The test schedule is shown in Figure 7. CTLA-4-humanized mice were C57BL / 6 mice into which the human CTLA-4 gene had been introduced.

[0101] Of the human CTLA-4 peptides used in the test, a peptide (hCTLA4) consisting of the amino acid sequence set forth in SEQ ID NO: 5 was identified as a peptide exhibiting the effect of inducing anti-CTLA-4 antibody production. Amino acids such as cysteine ​​were added to hCTLA4 to obtain a peptide (hCTLA4_V) consisting of the amino acid sequence set forth in SEQ ID NO: 4. Using hCTLA4_V as a short-chain peptide, an immunogenic composition (TT-AD-hCTLA4) was prepared by the method described above in <Preparation of Immunogenic Composition (2)>.

[0102] 8 shows the serum anti-human CTLA-4 antibody concentration in CTLA-4-humanized mice immunized with TT-AD-hCTLA4. It was confirmed that the serum anti-human CTLA-4 antibody concentration increased one week after the first boost.

[0103] Since the anti-CTLA-4 antibody production effect of TT-AD-hCTLA4 was comparable to that of TT-AD-mCTLA4-1 and TT-AD-mCTLA4-1 is presumed to have an antitumor effect, it was presumed that TT-AD-hCTLA4 also has an antitumor effect.

[0104] Example 4 Evaluation of the antitumor effect of immunogenic compositions using mouse PD-L1 peptides To identify mouse PD-L1 peptides that exhibit antitumor effects, immunogenic compositions were prepared using several types of mouse PD-L1 peptides with a length of 10 to 15 amino acids by the method described above in <Preparation of immunogenic compositions (1)>. C57BL / 6 mice were immunized with these immunogenic compositions, and then mouse colon cancer cells MC38 (10 ７ After transplantation, tumor size was measured over time. The test schedule is shown in Figure 9.

[0105] Of the mouse PD-L1 peptides used in the test, a peptide (mPDL1) consisting of the amino acid sequence set forth in SEQ ID NO: 7 was identified as a peptide exhibiting antitumor effects. Amino acids such as cysteine ​​were added to mPDL1 to obtain a peptide (mPDL1_V) consisting of the amino acid sequence set forth in SEQ ID NO: 6. Using mPDL1_V as a short-chain peptide, an immunogenic composition (TT-AD-mPDL1) was prepared by the method described above in <Preparation of Immunogenic Composition (1)>.

[0106] Figure 10 shows the serum anti-human PD-L1 antibody concentration in mice immunized with TT-AD-mPDL1. It was confirmed that the serum anti-human PD-L1 antibody concentration increased one week after the second boost. Figure 11 shows the change in tumor size over time in mice immunized with TT-AD-mPDL1. Tumor growth was suppressed in mice immunized with TT-AD-mPDL1 compared to when the control peptide was used. These results confirmed that TT-AD-mPDL1 has an anti-tumor effect.

[0107] Example 5 Evaluation of the antitumor effect of immunogenic compositions using human PD-L1 peptides To identify human PD-L1 peptides that exhibit antitumor effects, immunogenic compositions were prepared using several types of human PD-L1 peptides with a length of 10 to 15 amino acids by the method described above in <Preparation of immunogenic compositions (1)>. PD-L1-humanized C57BL / 6 mice were immunized with these immunogenic compositions, and then mouse colon cancer cells MC38 (10 ７ Cells) were transplanted into the mice. After transplantation, the tumor size was measured over time. The test schedule is shown in Figure 12. PD-L1 humanized C57BL / 6 mice are C57BL / 6 mice into which the human PD-L1 gene has been introduced.

[0108] Of the human PD-L1 peptides used in the test, a peptide (hPDL1) consisting of the amino acid sequence set forth in SEQ ID NO: 3 was identified as a peptide exhibiting antitumor effects. Amino acids such as cysteine ​​were added to hPDL1 to create a peptide (hPDL1_V) consisting of the amino acid sequence set forth in SEQ ID NO: 2. Using hPDL1_V as a short-chain peptide, an immunogenic composition (TT-AD-hPDL1) was prepared by the method described above in <Preparation of Immunogenic Composition (1)>.

[0109] Figure 13 shows the serum anti-human PD-L1 antibody concentration in immunized PD-L1 humanized mice. It was confirmed that the serum anti-human PD-L1 antibody concentration increased from two weeks after the primary immunization, and further increased one week after the first boost. Figure 14 shows the change in tumor size over time in immunized mice. Tumor growth was suppressed in mice immunized with TT-AD-hPDL1 compared to when the control peptide was used. These results confirmed that TT-AD-hPDL1 has an anti-tumor effect.

[0110] Example 6 Evaluation of persistence of antibody production after completion of immunization In CTLA4-humanized C57BL / 6 mice immunized with TT-AD-hCTLA4, the serum anti-human CTLA-4 antibody concentration after the final boost was measured to evaluate the persistence of antibody production.

[0111] The results are shown in Figure 15. Starting one week after the final boost, serum anti-human CTLA-4 antibody concentrations began to decrease, reaching nearly zero after eight weeks. These results confirmed that antibody production induced by TT-AD-hCTLA4 rapidly ceased upon termination of TT-AD-hCTLA4 administration.

[0112] Example 7 Evaluation of the number of amino groups in aminopolysaccharides Materials Tetanus toxoid (TT) 500 kDa aminodextran (AD) (400 amino groups / polymer) (AD500x400, Fina Biosolutions) (hereinafter also referred to as "AD400") 500 kDa aminodextran (AD) (50 amino groups / polymer) (AD500x50, Fina Biosolutions) (hereinafter also referred to as "AD50") 500 kDa aminodextran (AD) (25 amino groups / polymer) (AD500x250, Fina Biosolutions) (hereinafter also referred to as "AD25") Human PD-L1 peptide (hPDL1_V)

[0113] To evaluate the number of amino groups required per molecule in the aminopolysaccharide, immunogenic compositions were prepared using AD400, AD50, or AD25 as the aminopolysaccharide by the method described above in <Preparation of immunogenic compositions (1)>. PD-L1-humanized C57BL / 6 mice were immunized with these immunogenic compositions. Immunization was performed according to the test schedule shown in Figure 12.

[0114] Figure 16 shows the serum anti-human PD-L1 antibody concentration in immunized PD-L1 humanized mice. The serum anti-human PD-L1 antibody concentration increased when AD400 was used. When AD50 and AD25 were used, no increase in serum anti-human PD-L1 antibody concentration was confirmed. From these results, it was thought that in order to produce antibodies, it is necessary for each aminopolysaccharide molecule to contain at least about 100 amino groups.

[0115] <Preparation of Immunogenic Composition (3)> <Short-chain peptides: human PD-L1, mouse IgE> (Materials) Tetanus toxoid (TT) 500 kDa aminodextran (AD) (400 amino groups / polymer) (AD500 x 400, Fina Biosolutions) (AD400) 500 kDa aminodextran (AD) (50 amino groups / polymer) (AD500 x 50, Fina Biosolutions) (AD50) 500 kDa aminodextran (AD) (25 amino groups / polymer) (AD500 x 250, Fina Biosolutions) (AD25) Human PD-L1 peptide (hPDL1_V) Mouse PD-L1 peptide (mPDL1_V) Mouse IgE peptide 1 (mIgE-1_V) ([H]CPDHEPRGVITGSC[OH]; SEQ ID NO: 12), mouse IgE-derived portion: CPDHEPRGVIT (SEQ ID NO: 13) Mouse IgE peptide 2 (mIgE-2_V) ([H]CGSPRGVITYLGSC[OH]; SEQ ID NO: 14), mouse IgE-derived portion: PRGVITYL (SEQ ID NO: 15)

[0116] (Conjugation of short peptides to aminodextran and tetanus toxoid) (1) Dissolve 1.5 mg of tetanus toxoid (TT) in 1.5 mL of conjugation buffer (50 mM Na ２ HPO ４ , 50 mM NaH ２ P.O. ４, 150 mM NaCl, 10 mM EDTA, pH 7.4) to prepare a TT solution. (2) 0.4 mg of aminodextran (AD) was dissolved in 800 μL of conjugation buffer to prepare an AD solution. (3) 1.2 mg of sulfo-SMCC (Thermo Fisher Scientific) was dissolved in 250 μL of MilliQ water to prepare a sulfo-SMCC aqueous solution. (4) 60 μL and 40 μL of sulfo-SMCC aqueous solution were added to the TT solution and AD solution, respectively, and the mixture was incubated at room temperature for 1.5 hours. This introduced maleimide groups to the amino groups in the TT and AD. (5) Free sulfo-SMCC was removed by gel filtration using a Sephadex G-25 desalting column. (5) TCEP gel (Pierce ＴＭ The buffer in Immobilized TCEP Disulfide Reducing Gel (Thermo Fisher Scientific) was replaced with conjugation buffer. (6) The short-chain peptide was dissolved in conjugation buffer to prepare a 1 mg / mL short-chain peptide solution. The short-chain peptide solution was mixed with an equal amount of TCEP gel and incubated at room temperature for 1.5 hours using a rotator to reduce the cysteines at the ends of the short-chain peptides. After incubation, the reaction solution was centrifuged (500 × g, 5 minutes) to obtain the supernatant. (7) TT and the short-chain peptide in a 1 / 10 volume (weight ratio) and AD in a 1 / 5 to 1 / 5 volume (weight ratio) of the short-chain peptide were mixed and incubated overnight at room temperature. (8) Free short-chain peptides were removed by dialysis.

[0117] Immunogenic compositions prepared using short peptides, human PD-L1 peptide (hPDL1_V), mouse PD-L1 peptide (mPDL1_V), mouse IgE peptide 1 (mIgE-1_V), and mouse IgE peptide 2 (mIgE-2_V), respectively, and mixing a conjugate of short peptides with TT and a conjugate of short peptides with AD400 are described below. The same description applies when AD50 or AD25 is used instead of AD400. hPDL1_V: TT-hPDL1 / AD400-hPDL1 mPDL1_V: TT-mPDL1 / AD400-mPDL1 mIgE-1_V: TT-mIgE-1 / AD400-mIgE-1 mIgE-2_V: TT-mIgE-2 / AD400-mIgE-2

[0118] Example 8 (Evaluation of anti-human PD-L1 antibody production by immunogenic composition using human PD-L1 peptide) An immunogenic composition (TT-hPDL1 / AD400-hPDL1) was prepared using hPDL1_V as the short peptide by the method described above in <Preparation of immunogenic composition (3)>. PD-L1-humanized C57BL / 6 mice were immunized with the immunogenic composition (TT-hPDL1 / AD400-hPDL1). Immunization was performed in the same manner as the test schedule shown in Figure 12.

[0119] The serum anti-human PD-L1 antibody concentrations in immunized PD-L1-humanized mice are shown in Figure 17. It was confirmed that the serum anti-human PD-L1 antibody concentrations increased one week after the first boost and further increased one week after the second boost.

[0120] Example 9 (Evaluation of anti-mouse PD-L1 antibody production by various immunogenic compositions using mouse PD-L1 peptide) Using mPDL1_V as a short peptide, the following immunogenic compositions were prepared by the method described in <Preparation of immunogenic compositions (3)> above. TT-mPDL1 / AD400-mPDL1 TT-mPDL1 / AD50-mPDL1 TT-mPDL1 / AD25-mPDL1 TT-mPDL1 / AD400 TT-mPDL1

[0121] C57BL / 6 mice were immunized with the immunogenic composition described above. The immunization was performed in accordance with the test schedule shown in FIG.

[0122] Figure 18 shows the serum anti-mouse PD-L1 antibody concentration in immunized mice two weeks after the first immunization. Production of anti-mouse PD-L1 antibodies in the serum was confirmed only in immunized mice with TT-mPDL1 / AD400-mPDL1. Since antibody production could not be induced with an immunogenic composition (TT-mPDL1 / AD400) that mixed TT-mPDL1 and AD400, it was demonstrated that AD must be chemically bound to a short-chain peptide for antibody production.

[0123] Example 10 (Evaluation of anti-mouse IgE antibody production by immunogenic compositions using mouse IgE peptides) Using mIgE-1_V and mIgE-2_V as short peptides, immunogenic compositions (TT-hmIgE-1 / AD400-mIgE-1) and immunogenic compositions (TT-hmIgE-2 / AD400-mIgE-2) were prepared by the method described above in <Preparation of immunogenic compositions (3)>. C57BL / 6 mice were immunized with these immunogenic compositions. Immunization was performed in the same manner as the test schedule shown in Figure 9.

[0124] The serum anti-mouse IgE antibody concentrations in immunized mice are shown in Figure 19. It was confirmed that the serum anti-mouse IgE antibody concentrations increased one week after the second boost for all immunogenic compositions.

[0125] The present invention provides a highly immunogenic, inexpensive, and safe immunogenic carrier, and an immunogenic composition using the immunogenic carrier. Also provided are a pharmaceutical composition for treating or preventing cancer by enhancing cancer immunity, a pharmaceutical composition for treating or preventing obesity that can suppress the accumulation of neutral fat, and a pharmaceutical composition for treating or preventing allergic diseases that can inactivate the action of IgE.

[0126] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

[0127] SP: short-chain peptide AS: aminopolysaccharide CP: carrier protein CPP: carrier protein-derived peptide DC: dendritic cell Th: helper T cell TCR: T cell receptor TP: target protein Ab1: antibody against target protein Ab2: antibody against carrier protein BCR1: B cell receptor capable of binding to short-chain peptide BCR2: B cell receptor capable of binding to carrier protein B1: B cell having BCR1 B2: B cell having BCR2

Claims

1. An immunogenic carrier comprising an aminopolysaccharide containing 100 or more amino groups per molecule and a protein.

2. The immunogenic carrier according to claim 1, wherein the aminopolysaccharide and the protein are chemically bonded to each other.

3. The immunogenic carrier according to claim 1, wherein the aminopolysaccharide and the protein are not chemically bound to each other.

4. The immunogenic carrier according to any one of claims 1 to 3, wherein the protein is a bacterial toxoid.

5. The immunogenic carrier of claim 4, wherein said bacterial toxoid is tetanus toxoid.

6. The immunogenic carrier according to any one of claims 1 to 3, wherein the aminopolysaccharide is an aminodextran.

7. An immunogenic composition comprising the immunogenic carrier according to any one of claims 1 to 3 and a short peptide.

8. The immunogenic composition of claim 7, wherein the short peptide is chemically bound to the immunogenic carrier.

9. The immunogenic composition of claim 8, wherein the short peptides are chemically bound to the aminopolysaccharide.

10. The immunogenic composition of claim 9, wherein the short peptide is chemically bound to the protein.

11. The immunogenic composition of claim 7, wherein the short peptide comprises an amino acid sequence derived from a protein selected from the group consisting of a tumor antigen protein, an immune checkpoint protein, and immunoglobulin E.

12. The immunogenic composition of claim 11, wherein the short peptide comprises an amino acid sequence derived from PD-L1, an amino acid sequence derived from CTLA-4, or an amino acid sequence derived from immunoglobulin E.

13. The immunogenic composition according to claim 12, wherein the short peptide is a peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 or 5.

14. The immunogenic composition according to claim 11, which is used for treating or preventing a tumor or an atopic disease.