Vaccine composition for inducing anti-IgE antibodies

Abstract

The present invention provides a vaccine composition capable of inducing the production of antibodies against IgE, comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, wherein the B cell receptor antigen peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22.

Description

[Technical field]

[0001] The present invention relates to a vaccine composition, and more particularly to a vaccine composition capable of inducing antibodies against IgE in the body. [Background technology]

[0002] IgE is a member of the immunoglobulin family that mediates allergic responses in type I allergic diseases (e.g., asthma, allergic rhinitis, urticaria, etc.). The mechanism by which IgE causes type I allergic diseases is briefly as follows: IgE binds to the high-affinity IgE receptor (FcεR1) present on the cell surface of mast cells and basophils, and when an allergen binds to the IgE to form a cross-link, the mast cells and basophils release chemical mediators including histamine. The released chemical mediators increase vascular dilation and vascular permeability, resulting in various allergic symptoms.

[0003] Omalizumab (trade name Xolair (registered trademark)), an anti-IgE monoclonal antibody, is commercially available as one of the therapeutic agents for type I allergic diseases. Omalizumab specifically binds to free IgE in the blood, and by removing it, it reduces the frequency of binding between FcεR1 on the surface of mast cells and IgE, thereby suppressing the activation of mast cells.

[0004] Omalizumab has a certain effect on severe asthma and is one of the effective therapeutic drugs for allergic diseases, but there are still some areas for improvement. For example, omalizumab is a humanized mouse monoclonal antibody, and immunological reactions cannot be completely avoided when used in human patients. In addition, antibody drugs are expensive, and the cost of treatment with omalizumab is said to be 15,000 to 44,000 US dollars per patient per year. Furthermore, antibody drugs have disadvantages such as limited administration methods. Against this background, there is a strong demand for the development of a safer and less expensive method of treating allergic diseases.

[0005] In order to overcome such disadvantages, the development of alternative means, such as antigen peptides that can induce anti-IgE antibodies by administration to a living body, is being actively pursued. For example, Patent Document 1 discloses an IgE immunogen construct that can induce anti-IgE antibodies. However, there is a constant demand for the development of IgE immunogens that can efficiently induce anti-IgE antibodies. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2010067286 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above-mentioned background, an objective of the present invention is to provide a novel IgE immunogen capable of inducing anti-IgE antibodies extremely efficiently. [Means for solving the problem]

[0008] As a result of intensive research into the above-mentioned problems, the present inventors have found that a complex of a T cell receptor antigen peptide having a specific amino acid sequence and a B cell receptor antigen peptide having a specific amino acid sequence in the Cε3 region of the Fc region of IgE can induce anti-IgE neutralizing antibodies extremely efficiently in vivo. The complex discovered by the present inventors had excellent IgE immunogenicity and was able to efficiently induce antibodies with high IgE neutralizing activity. Based on these findings, the present inventors have further pursued research and have completed the present invention.

[0009] That is, the present invention is as follows. [1] A vaccine composition capable of inducing the production of antibodies against IgE, comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, wherein the B cell receptor antigen peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22. [2] The vaccine composition described in [1], wherein the T cell receptor antigen peptide comprises the amino acid sequence represented by sequence number 1. [3] The vaccine composition according to [1] or [2], wherein the complex is bound between the C-terminus of the T cell receptor antigen peptide and the N-terminus of the B cell receptor antigen peptide. [4] The vaccine composition according to any one of [1] to [3], wherein the T cell receptor antigen peptide and the B cell receptor antigen peptide are linked via a linker. [5] The vaccine composition according to any one of [1] to [4], for treating or preventing a disease accompanied by hypersecretion of IgE. [6] The vaccine composition according to [5], wherein the disease accompanied by excessive secretion of IgE is at least one selected from the group consisting of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and anaphylactic hypersensitivity. [A-1] A method for treating or preventing a disease accompanied by excessive secretion of IgE in a subject, comprising administering to the subject a vaccine composition capable of inducing production of antibodies against IgE, the vaccine composition comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, wherein the B cell receptor antigen peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22. [A-2] The method described in [A-1], wherein the T cell receptor antigen peptide comprises the amino acid sequence represented by sequence number 1. [A-3] The method described in [A-1] or [A-2], wherein the complex is bound between the C-terminus of the T cell receptor antigen peptide and the N-terminus of the B cell receptor antigen peptide. [A-4] The method according to any one of [A-1] to [A-3], wherein the T cell receptor antigen peptide and the B cell receptor antigen peptide are linked via a linker. [A-5] The method according to any one of [A-1] to [A-4], wherein the disease accompanied by excessive secretion of IgE is at least one selected from the group consisting of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and anaphylactic hypersensitivity. [B-1] A vaccine composition capable of inducing the production of antibodies against IgE, comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, for use in the treatment or prevention of a disease accompanied by excessive secretion of IgE, wherein the B cell receptor antigen peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22. [B-2] A vaccine composition for use according to [B-1], wherein the T cell receptor antigen peptide comprises the amino acid sequence represented by SEQ ID NO:1. [B-3] A vaccine composition for use according to [B-1] or [B-2], wherein the complex is bound between the C-terminus of the T cell receptor antigen peptide and the N-terminus of the B cell receptor antigen peptide. [B-4] The vaccine composition for use according to any one of [B-1] to [B-3], wherein the T cell receptor antigen peptide and the B cell receptor antigen peptide are linked via a linker. [B-5] The vaccine composition for use according to any one of [B-1] to [B-4], wherein the disease accompanied by excessive secretion of IgE is at least one selected from the group consisting of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and anaphylactic hypersensitivity. [C-1] Use of a vaccine composition capable of inducing the production of antibodies against IgE, comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, in the manufacture of a pharmaceutical for treating or preventing a disease accompanied by excessive secretion of IgE, wherein the B cell receptor antigen peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22. [C-2] The use described in [C-1], wherein the T cell receptor antigen peptide comprises the amino acid sequence represented by sequence number 1. [C-3] The use according to [C-1] or [C-2], wherein the complex is bound between the C-terminus of the T cell receptor antigen peptide and the N-terminus of the B cell receptor antigen peptide. [C-4] The use according to any one of [C-1] to [C-3], wherein the T cell receptor antigen peptide and the B cell receptor antigen peptide are linked via a linker. [C-5] The use according to any one of [C-1] to [C-4], wherein the disease accompanied by excessive secretion of IgE is at least one selected from the group consisting of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and anaphylactic hypersensitivity. Effect of the Invention

[0010] According to the present invention, it is possible to induce the production of anti-IgE antibodies in the body very efficiently, thereby enabling inexpensive and safe treatment and / or prevention of diseases accompanied by excessive secretion of IgE, such as type I allergic diseases. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 shows the results of measuring the antibody titer against IgE synthetic peptide in antisera derived from mice administered AJP001 conjugated peptides having any of the B cell epitope sequences of SEQ ID NOs: 2 to 34 by ELISA, and the results of measuring the antibody titer against human IgE in antisera derived from mice administered AJP001 conjugated peptides having any of the B cell epitope sequences of SEQ ID NOs: 16 to 28 by ELISA. (GMT±95%CI (N=4)) [Diagram 2] FIG. 2 shows the results of evaluating the neutralizing activity (inhibition of Luciferase expression, Mean±SD (Triplicate)) of antisera from rats administered with AJP001 conjugated peptides having any of the B cell epitope sequences of SEQ ID NOs: 16 and 20 to 24. [Diagram 3] FIG. 3 shows antibody titers (GMT±95% CI, (N=8)) in mouse serum against each AJP001 conjugated peptide or human IgE when AJP001 conjugated peptide having the B cell epitope sequence of SEQ ID NO: 20 or 21 was administered to humanized IgE / FcεR1 Tg mice. [Figure 4] FIG. 4 is a diagram showing the complex concentration (Mean±SE (N=7 or 8)) in the serum of humanized IgE / FcεR1 Tg mice when the mice were administered an AJP001 conjugated peptide having a B cell epitope sequence of SEQ ID NO: 20 or 21. [Diagram 5] FIG. 5 shows the number of nose-scratching behaviors in humanized IgE / FcεR1 Tg mice when the mice were administered an AJP001 conjugated peptide having a B cell epitope sequence of SEQ ID NO: 20 or 21 (Mean±SE (N=8), **: p< 0.01 vs OVA group (t test), ♯: p< 0.01 vs OVA+Adjuvant group (t test)), and a correlation diagram (Spearman's rank correlation coefficient) between the number of nose-scratching behaviors and the complex concentration. [Figure 6]FIG. 6 shows serum OVA-hIgE concentrations (Min. to Max. (N=4-8)) and NALF Eotaxin concentrations (Min. to Max. (N=4-8), *: P<0.05 vs. OVA+Adjuvant group (t test)) in humanized IgE / FcεR1 Tg mice when the mice were administered an AJP001 conjugated peptide having the B cell epitope sequence of SEQ ID NO: 20 or 21. [Figure 7] FIG. 7 shows histopathological examination and eosinophil infiltration in the nasal tissue of humanized IgE / FcεR1 Tg mice when the mice were administered an AJP001 conjugated peptide having a B cell epitope sequence of SEQ ID NO: 20 or 21 (Mean±SE (N=4-8), **: P<0.01 vs. OVA+Adjuvant group (t test)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below.

[0013] 1. Vaccine Composition The present invention provides a vaccine composition capable of inducing the production of antibodies against IgE, comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, wherein the B cell receptor antigen peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22 (hereinafter sometimes referred to as the "vaccine composition of the present invention").

[0014] In the peptides described herein, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide notation. The peptide conjugate contained as an active ingredient in the vaccine composition of the present invention has a carboxyl group (-COOH) at the C-terminus, a carboxylate (-COOH) at the C-terminus, and a carboxyl group (-COOH) at the C-terminus. - ), amide (-CONH2) or ester (-COOR).

[0015] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, and n-butyl. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0016] When the peptide conjugate has a carboxyl group (or carboxylate) other than at the C-terminus, the peptide conjugate of the present invention also includes those in which the carboxyl group is amidated or esterified. In this case, the ester may be, for example, the C-terminus ester described above.

[0017] Furthermore, the peptide complex may have a protecting group (e.g., a C group such as a formyl group or an acetyl group) at the amino group of the N-terminal amino acid residue. 1-6 Alkanoyl C 1-6 those in which the amino group of the N-terminal amino acid residue is acetylated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by suitable protecting groups (e.g., C groups such as formyl group, acetyl group, etc.); 1-6 Alkanoyl group etc. 1-6 In one embodiment, the peptide conjugate is protected with an acetyl group (such as an acyl group) at the amino acid residue at the N-terminus and / or an amidated carboxyl group at the C-terminus.

[0018] The peptide complex contained as an active ingredient in the vaccine composition of the present invention contains a B cell receptor antigen peptide as a part thereof. Here, the B cell receptor is a receptor expressed on the surface of B cells. B cells stimulated by the antigen peptide proliferate and secrete the B cell receptor as an antibody against the antigen peptide.

[0019] The B cell receptor antigen peptide in the vaccine composition of the present invention comprises an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22. Specifically, the amino acid sequences represented by SEQ ID NOs: 20 to 22 are as follows:

[0020] GKPVNHSTRKEEKQRNGT (SEQ ID NO:20) SGKPVNHSTRKEEKQRNGT (SEQ ID NO:21) ASGKPVNHSTRKEEKQRNGT (SEQ ID NO:22)

[0021] In one embodiment of the present invention, the B cell receptor antigen peptide can comprise or consist of an amino acid sequence represented by any one of SEQ ID NOs:20-22.

[0022] Part of the complex contained in the vaccine composition of the present invention is a T cell receptor antigen peptide. The T cell receptor antigen peptide is not particularly limited as long as it is an antigen peptide that forms a complex with MHC class II, is recognized by the CD3 / TCR complex and CD4, and transmits a signal into CD3-positive cells. MHC class II includes, for example, HLA-DR, HLA-DQ, and HLA-DP in humans, and H-2A or H-2B in mice, each of which is a dimer consisting of an α chain and a β chain (for example, HLA-DR is an α chain, HLA-DRA, and a β chain, HLA-DRB1), and is preferably HLA-DR, HLA-DQ, or HLA-DP.

[0023] In the vaccine composition of the present invention, the T cell receptor antigen peptide is not particularly limited as long as it contains an epitope sequence that can be presented on the MHC class II molecule of an antigen-presenting cell to activate a helper T cell. For example, in addition to the AJP001 peptide developed by the present inventors (ELKLIFLHRLKRLRKRLKRK (SEQ ID NO: 1), see WO2016 / 047763 for details), UBITh peptides (UBITh (registered trademark) 1: ISITEIKGVIVHRIETILF (SEQ ID NO: 35), UBITh (registered trademark) 2: KKKIITITRIITIITTID (SEQ ID NO: 36), UBITh (registered trademark) 3: ISISEIKGVIVHKIETILF (SEQ ID NO: 37), ISITEIRTVIVTRIETILF (SEQ ID NO: 38), see US patent no. 9,102,752 for details), Tetanous toxisoid peptide (TTaa830-843 peptide: QYIKANSKFIGITE (SEQ ID NO: 39)), etc. can be used. AJP001 is particularly preferred as a T cell receptor antigen peptide in the present invention because it can activate the innate immune system by inducing the secretion of IL-β1 and IL-18 through activation of the NLRP3 inflammasome and by inducing the production of TNF-α and IL-6 through the NF-κB pathway.

[0024] In the vaccine composition of the present invention, the T cell receptor antigen peptide and the B cell receptor antigen peptide are bound to each other to form a complex. The binding may be between the end of one peptide chain and the end of the other peptide chain, between an amino acid side chain of one peptide and the end of the other peptide chain, or between both amino acid side chains, but is preferably between the end of one peptide chain and the end of the other peptide chain, more preferably between the C-terminus of one peptide chain and the N-terminus of the other peptide chain, and even more preferably between the C-terminus of the T cell receptor antigen peptide and the N-terminus of the B cell receptor antigen peptide. When the end of one peptide chain and the end of the other peptide chain are linked, the terminal amino acids of both may be directly linked by a peptide bond, or may be linked via a linker (also referred to as "spacer" in the present specification). The linker is not particularly limited as long as it can link the T cell receptor antigen peptide and the B cell receptor antigen peptide and can be taken up into an antigen-presenting cell to present a helper T cell epitope in the T cell receptor antigen peptide on a free MHC class II molecule. For example, amino acids other than α-amino acids such as ε-aminocaproic acid, β-aminoalanine, γ-aminobutyric acid, 7-aminoheptanoic acid, 12-aminolauric acid, and p-aminobenzoic acid can be used. In addition, L-amino acids (e.g., glutamic acid, cysteine, and lysine) present in natural proteins and their D-amino acids can also be used. In a preferred embodiment, the amino acid linker is ε-aminocaproic acid. Alternatively, a peptide linker consisting of any 2 to 15 amino acids can be used. Examples include, but are not limited to, a G linker, which is a peptide linker consisting of glycine (Gly) or methylated glycine (MeG), and a GS linker, which is a peptide linker consisting of Gly or MeG and Ser. In another embodiment, a PEG linker containing polyethylene glycol (PEG) or a polyethylene glycol derivative can be used. A PEG linker further containing one or more selected from glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg), and lysine (Lys) can also be used.

[0025] In one embodiment, the complex in which the B cell receptor antigen peptide and the T cell receptor antigen peptide are linked may further contain additional amino acids. Such addition of amino acids is permitted as long as the desired effect of the vaccine composition of the present invention is obtained. The added amino acid sequence is not particularly limited, but may be, for example, a tag for facilitating detection or purification of the complex. Examples of tags include Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (e.g., GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. The position at which the amino acid sequence is added is not particularly limited, but is preferably the N-terminus and / or C-terminus of the complex. In another embodiment, the complex in the vaccine composition of the present invention may be bound to a functional molecule other than the above-mentioned tag. Such a functional molecule is not particularly limited as long as the vaccine composition of the present invention exerts the desired effect. Examples of such a functional molecule include, but are not limited to, a molecule having a function of suppressing the decomposition of the complex when the vaccine composition of the present invention is administered to a living body.

[0026] The conjugate in the vaccine composition of the present invention can be produced by solid-phase synthesis (Fmoc method and Boc method) or liquid-phase synthesis according to known general peptide synthesis protocols. In the case of a conjugate in which the B cell receptor antigen peptide and the T cell receptor antigen peptide are linked directly or via an amino acid or peptide linker, the entire conjugate can be synthesized at once. Alternatively, the B cell receptor antigen peptide and the T cell receptor antigen peptide may be synthesized separately, and then the two peptides may be linked directly or via a linker.

[0027] In one embodiment, the peptide complex in the vaccine composition of the present invention may be conjugated with a carrier protein to enhance its immunogenicity. A carrier protein is generally a substance that binds to a molecule (hapten) that is not immunogenic due to its small molecular weight to impart immunogenicity, and some of these are known in the art. Examples of carrier proteins include bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), thyroglobulin (TG), diphtheria toxin (CRM197) detoxified by replacing a portion of the amino acid of diphtheria toxin, immunoglobulin, and the like. The carrier protein can be conjugated to the N-terminus or C-terminus of the complex in the vaccine composition of the present invention. As a method for conjugation, a cysteine ​​residue is introduced into the antigen peptide of the present invention, and the peptide is conjugated to the amino group of the carrier protein via the SH group, which is the side chain of the cysteine ​​(MBS method). Conjugation can also be achieved by binding amino groups, such as the ε-amino group or α-amino group of a lysine residue in a protein (glutaraldehyde method).

[0028] In one embodiment, the vaccine composition of the present invention may further contain an adjuvant that is pharma- ceutical acceptable and compatible with the active ingredient. Adjuvants are generally substances that non-specifically enhance the immune response of a host, and many adjuvants are known in the art. The adjuvant used in the vaccine composition of the present invention is not particularly limited as long as it can non-specifically enhance the immune response, and examples thereof include aluminum salts (Alum), alum, CpG oligodeoxynucleotide, dsRNA, Montanide (trademark of Seppic), squalane, saponin, and the like.

[0029] The vaccine composition of the present invention may be provided as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier in addition to the complex of the T cell receptor antigen peptide and the B cell receptor antigen peptide.

[0030] Pharmaceutically acceptable carriers may be appropriately selected depending on the dosage form, and examples include, but are not limited to, excipients such as sucrose, starch, etc., binders such as cellulose, methylcellulose, etc., disintegrants such as starch, carboxymethylcellulose, etc., lubricants such as magnesium stearate, fragrances such as citric acid, menthol, etc., preservatives such as sodium benzoate, sodium bisulfite, etc., stabilizers such as sodium citrate, suspending agents such as methylcellulose, polyvinylpyrrolidone, etc., dispersing agents such as surfactants, diluents such as water, physiological saline, etc., base waxes, etc.

[0031] The vaccine composition of the present invention can be administered orally or parenterally to a mammal. Since the complex of the T cell receptor antigen peptide and the B cell receptor antigen peptide can be decomposed in the stomach, parenteral administration is preferable. Examples of preparations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions, etc. Examples of preparations suitable for parenteral administration (e.g., subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, isotonicity agents, etc. Also, examples include aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. The preparations can be sealed in containers such as ampoules and vials in unit doses or multiple doses. In addition, the active ingredient and the pharma-ceutical acceptable carrier can be freeze-dried and stored in a state in which they only need to be dissolved or suspended in a suitable sterile vehicle immediately before use.

[0032] The content of the active ingredient (i.e., peptide complex) in the vaccine composition is usually about 0.001 to 100% by weight, preferably about 0.05 to 99% by weight, and more preferably about 0.1 to 90% by weight of the total composition, but is not limited to these.

[0033] The subject to which the vaccine composition of the present invention is administered is not particularly limited, so long as it is a mammalian animal that can suffer from a disease whose pathology may worsen due to excessive secretion of IgE (hereinafter, sometimes referred to as a "disease associated with excessive secretion of IgE"). Examples of such mammalian animals include rodents such as mice, pets such as dogs and cats, livestock such as pigs, horses, and cows, and primates such as humans, monkeys, orangutans, and chimpanzees, with humans being particularly preferred.

[0034] The dosage of the vaccine composition of the present invention will vary depending on the recipient, administration method, administration form, etc., but typically, per adult, the active ingredient, a complex of T cell receptor antigen peptide and B cell receptor antigen peptide, is administered in the range of 1 μg to 300,000 μg per dose, preferably in the range of 20 μg to 30,000 μg, two to three times over a period of four to twelve weeks, and an additional dose can be administered each time the antibody titer decreases.

[0035] Diseases that can be treated or prevented by the vaccine composition of the present invention are those associated with the hypersecretion of IgE, including, but not limited to, asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and anaphylactic hypersensitivity.

[0036] In one embodiment, the vaccine composition of the present invention may be used in combination with an existing therapeutic agent for a disease accompanied by excessive secretion of IgE. For example, in the treatment of an allergic disease, treatment can be started with a highly fast-acting antibody drug (e.g., omalizumab, etc.) during the active phase of the disease, and administration of the vaccine composition of the present invention can be started during the remission phase, thereby achieving both the patient's QOL and treatment costs. In addition, the vaccine composition of the present invention and an existing therapeutic agent can be used simultaneously.

[0037] In this specification, "treatment" of a disease may include not only curing the disease, but also alleviating the disease and improving the severity of the disease.

[0038] In addition, the term "prevention" of a disease as used herein includes not only preventing the onset of a disease but also delaying the onset of a disease. In addition, the term "prevention" of a disease as used herein may also include preventing the recurrence of the disease after treatment or delaying the recurrence of the disease after treatment.

[0039] In addition, the term "vaccine composition" as used herein may be alternatively referred to as "pharmaceutical composition" or "medication."

[0040] 2. Method for treating or preventing a disease associated with hypersecretion of IgE The present invention also provides a method for treating or preventing a disease associated with hypersecretion of IgE (hereinafter sometimes referred to as the "method of the present invention"), which comprises administering a vaccine composition of the present invention to a subject suffering from or likely to suffer from a disease associated with hypersecretion of IgE.

[0041] In the method of the present invention, the subject to be treated or prevented, the conditions for administering the vaccine composition of the present invention, etc. are the same as those explained in "1. Vaccine composition of the present invention."

[0042] In one aspect, the present invention provides a method for inducing production of antibodies against IgE in a subject, comprising administering the vaccine composition of the present invention to the subject. The subject, administration conditions of the vaccine composition of the present invention, and the like in such a method are the same as those described in "1. Vaccine composition of the present invention."

[0043] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples in any way. EXAMPLES

[0044] Peptide synthesis (Fmoc method) Protected peptide resin was synthesized by Fmoc method using a fully automated solid-phase synthesizer according to the method described in Experimental Chemistry Course 16, Synthesis of Organic Compounds IV, 5th Edition. Trifluoroacetic acid (TFA) and a scavenger (a mixture of thioanisole, 2,2'-(ethylenedioxy)diethanethiol, m-cresol, triisopropylsilane, and water) were added to the obtained protected peptide resin, which was cut off from the resin and deprotected to obtain a crude peptide. This crude peptide was purified by gradient elution using a reversed-phase HPLC column with a 0.1% TFA-H20 / CH3CN system. Fractions containing the target product were collected and lyophilized to obtain the target peptide.

[0045] HPLC analysis of peptides The purity of the synthesized peptide was measured using an HPLC system under the following analytical conditions. HPLC model: Shimadzu LCLC-20ADXR Measurement wavelength: 220 nm Flow rate: 0.31 mL per minute Column: Inertsil ODS-3, 2.1m x 250mm, 5micron Column temperature: room temperature Mobile phase A: 0.1% trifluoroacetic acid in water Mobile phase B: Acetonitrile Gradient conditions: A linear gradient of mobile phase B from 5% to 80% in 30 minutes (5→80% buffer B in 30 min).

[0046] Mass spectrometry of peptides The mass of the synthesized peptide was measured by MALDI-TOF-MS under the following analytical conditions. MALDI-TOF-MS model: Bruker autoflex speed Matrix: 2,5-Dihydroxybenzoic acid Dissolving solution: A mixture of 0.1% trifluoroacetic acid and acetonitrile

[0047] [Example 1] Immunogenicity evaluation test in rats using AJP001 conjugated peptide (human IgE) The human IgE epitope peptides shown in Table 2 (SEQ ID NOs: 2 to 34) were selected as B cell antigens, and conjugated complexes (AJP001 conjugate peptides) were prepared using the T cell antigen AJP001 (Table 1, SEQ ID NO: 1) and ε-aminocaproic acid (sometimes referred to as "Ahx") as a spacer (production was outsourced to Toray Research Center, Inc. or Peptide Institute, Inc.). In this specification, the AJP001 conjugate peptide having an amino acid sequence represented by SEQ ID NO: "X" as the B cell epitope will be referred to as "AJP001 conjugate peptide (SEQ ID NO: X)" and the like.

[0048] [Table 1]

[0049] [Table 2]

[0050] AJP001 conjugate peptides having any of the B cell epitope sequences of SEQ ID NO:2 to 34 were dissolved in physiological saline, mixed with 2% Alhydrogel (invivogen, 0.3 mg / body) and K3 Et-Free (Gene Design, 0.1 mg / body), and subcutaneously administered to JCL:Wistar rats (female, 7 weeks old, N=4) at 0.5 mg / body, 3 times at 2-week intervals. Blood was collected before administration and 6 weeks after the first administration, and antibody titers against each epitope sequence and human IgE were measured by ELISA. Specifically, a 96-well plate on which epitope peptides dissolved at 10 μg / mL in carbonate buffer were immobilized was blocked with 5% skim milk / PBS, and serum serially diluted with 5% skim milk / PBS was added and left to stand overnight at 4°C. After washing the wells with PBS-T, HRP-labeled anti-rat IgG antibody (BETHYL) diluted with 5% skim milk / PBS was added and shaken at room temperature for 3 hours. After washing the wells with PBS-T, TMB solution (SIGMA) was added and left to stand for 30 minutes in the dark, 0.1M H2SO4 (Kanto Chemical) was added to stop the reaction, and the absorbance at 450 nm was measured with a plate reader. The serum dilution ratio at which the absorbance was 1 / 2 the maximum (OD=1.75) was defined as the antibody titer, and the geometric mean titer (GMT) was calculated from the individual values. The figure shows the geometric mean titer (GMT) ± 95% confidence interval (95% CI). The antibody titer against human IgE was measured by ELISA in the same manner as above, using human IgE full length protein (abcam) as the solid-phase antigen.

[0051] The antibody titers against each AJP001 conjugated peptide and human IgE are shown in Figure 1. As a result, a significant increase in human IgE antibody titer was confirmed in the antisera of rats administered with the AJP001 conjugated peptides having the B cell epitope sequences of SEQ ID NOs: 17 to 27.

[0052] [Example 2] Evaluation of neutralizing activity of AJP001 conjugated peptide (human IgE) using rat antiserum The neutralizing activity of anti-human IgE antibodies produced by the AJP001 conjugate peptide was evaluated using serum 6 weeks after the first administration in Example 1. A stable expression cell line (RBL-2H3 / FCER1A+NFAT NLuc cell) was used, which was established by introducing human FcεRIα and NFAT-RE-Luciferase genes into RBL-2H3. When human IgE is added to these cells, they bind to FcεRIα, and then stimulate with anti-human IgE antibody to activate NFAT and express a reporter gene (Luciferase). The inhibition of Luciferase expression upon stimulation with human IgE was used as an index for evaluation. Specifically, for SEQ ID NOs: 16 and 20 to 24, pooled serum (400 μL / individual, N=4) was prepared, inactivated (56°C, reacted for 30 minutes), and then an equal amount of ammonium sulfate (FUJIFILM Wako Pure Chemical) was added and reacted at room temperature for 30 minutes while mixing by inversion. The mixture was then centrifuged at 3,000 × g for 20 minutes at 4°C, and the precipitate was dissolved in PBS. Serum IgG antibodies (including anti-IgE antibodies) were purified using a Protein G HP spin trap (Cytiva), an Ab buffer kit (Cytiva), and Amicon ultra-0.5 centrifugal filter devices (100K) (Millipore). Next, the purified IgG (final concentration 1.2-500 μg / mL) and human IgE (final concentration 0.6 μg / mL) were added to the culture medium (1% P / S, 400 μg / mL G418, 500 μg / mL hygromycin B, 10% FBS-containing DMEM) and reacted at 37°C for 2 hours, and then added at 50 μL / well to cells (1.0 × 10^5 cells / 50 μL / well) seeded on a 96-Well white plate (ThermoFisher) and reacted at 37°C for 24 hours. In addition, untreated wells and control wells to which only human IgE was added were set up. After 24 hours, the plate was washed with the maintenance medium, and anti-human IgE antibody (final concentration 1 μg / mL, AQI) was added and reacted at 37°C for 4 hours. NanoGlo Luciferase Assay substrate was then added, reacted for 3 minutes, and the luminescence value was measured with a luminescence plate reader.The percentage of luminescence value relative to the control well was calculated from the luminescence values ​​of untreated wells (A), control wells (B), and wells (C) containing purified IgG derived from rat antisera administered with each AJP001 conjugated peptide using the following formula.

[0053] Based on the concentration of purified IgG added, a regression equation was calculated using a 4-parameter logistic model, and the 50% inhibitory concentration (IC50) was calculated from the regression equation. The neutralizing activity was expressed in stages based on the IC50 concentration (μg / mL) (+: 10^4 digits, ++: 10^3 digits, +++: 10^2 digits, when calculation was impossible, it is indicated as NC).

[0054] Percentage of luminescence value relative to control well (%) = 100 x [(CA) / (BA)]

[0055] The neutralizing activity against human IgE is shown in Figure 2. As a result, neutralizing activity (inhibition of luciferase expression) was confirmed in the antisera from rats administered with AJP001 conjugate peptides having the B cell epitope sequences of SEQ ID NOs: 20, 21, and 22.

[0056] [Example 3] Efficacy evaluation test of AJP001 conjugated peptide (human IgE) on OVA-induced allergic rhinitis model using humanized IgE / FcεR1 Tg mice AJP001 conjugate peptide having the B cell epitope sequence of SEQ ID NO: 20 or 21 was dissolved in physiological saline, mixed with 2% Alhydrogel (Invivogen, 0.3 mg / body) and K3 Et-Free (Gene Design, 0.1 mg / body), and subcutaneously administered to Humanized IgE / FcεR1 Tg mice (female, 10 weeks old, N=8 / group) at 0.5 mg / body, 3 times at 2-week intervals (Day 0, 14, 28). A positive control antihistamine (Bilastine, Taiho Pharmaceutical Co., Ltd.) was orally administered at 50 mg / kg on Day 56 to 66, 1 hour before OVA administration. In addition, an OVA+adjuvant group was set as a control group for the SEQ ID NO: 20 or 21 administration group, and an OVA group was set as a control group for the Bilastine administration group. The pathological model was created by administering OVA (containing Alum) at 100 μg / body (intraperitoneally) on Day 35 and 50 μg / body (subcutaneously) on Day 49, followed by intranasal administration of 50 μg of OVA on Days 56 to 66. Under these test conditions, serum was collected (Days 0, 14, 28, 35, 49, and 67), and the antibody titers against each AJP001 conjugate peptide and human IgE, the complex concentration of human IgE / anti-human IgE antibodies in the serum, and the OVA-specific human IgE concentration in the serum were measured by ELISA. In addition, video recording was performed 1 hour after intranasal administration on Days 55, 60, and 66, and the number of nose-scratching behaviors was evaluated. After euthanasia on Day 67, nasal lavage fluid (NALF) was collected, and the Eotaxin concentration in the NALF was measured by ELISA. Furthermore, nasal tissue was collected, Luna-stained specimens were prepared, and the number of eosinophil infiltrations was measured. Antibody titers were measured using the same procedures as in Example 1. Serum OVA-specific human IgE concentrations were measured using a Human Ovalbumin Specific IgE ELISA Kit (Finetest), and NALF Eotaxin concentrations were measured using a Mouse CCL11 / Eotaxin immunoassay kit (R&D).

[0057] The antibody titers against each AJP001 conjugated peptide and human IgE are shown in Figure 3, the serum complex concentrations in Figure 4, the correlation diagram between the number of nose-scratching behaviors and the complex concentrations in Figure 5, the serum OVA-hIgE concentrations and the Eotaxin concentrations in NALF in Figure 6, and the histopathological examination and the degree of eosinophil infiltration in Figure 7.

[0058] As a result, in the group administered with SEQ ID NO: 20 or 21, the antibody titer increased over time, and the number of nose scratchings and eosinophil infiltration in the nasal tissue were significantly suppressed. In addition, the eotaxin concentration in NALF and the OVA-specific human IgE concentration in serum also tended to be low. These results suggested that the antibodies induced in the group administered with SEQ ID NO: 20 or 21 could suppress rhinitis symptoms by binding to human IgE and inhibiting IgE / FcεR1 binding. On the other hand, the antihistamine (bilastine) showed the same effectiveness in the number of nose scratchings as the group administered with SEQ ID NO: 20 or 21, but other parameters did not change. This suggests that the IgE antibody-inducing peptide and the antihistamine suppress rhinitis symptoms by different mechanisms of action. [Industrial Applicability]

[0059] INDUSTRIAL APPLICABILITY The present invention enables the production at low cost of peptide vaccines for the treatment and / or prevention of diseases accompanied by excessive secretion of IgE, such as type I allergic diseases, and is therefore extremely useful in the field of pharmaceutical production.

[0060] This application is based on patent application No. 2023-191836 filed in Japan (filing date: November 9, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A vaccine composition capable of inducing the production of antibodies against IgE, comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, wherein the B cell receptor antigen peptide consists of an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22, wherein the T cell receptor antigen peptide has the amino acid sequence represented by SEQ ID NO:

1.

2. 2. The vaccine composition of claim 1, wherein the conjugate is linked between the C-terminus of the T cell receptor antigen peptide and the N-terminus of the B cell receptor antigen peptide.

3. 3. The vaccine composition according to claim 1 or 2, wherein the T cell receptor antigen peptide and the B cell receptor antigen peptide are linked via a linker.

4. The vaccine composition according to claim 1 or 2 for treating or preventing a disease accompanied by hypersecretion of IgE.

5. The vaccine composition according to claim 4, wherein the disease accompanied by hypersecretion of IgE is at least one selected from the group consisting of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and anaphylactic hypersensitivity.

6. A peptide having the following amino acid sequence: Ac-ELKLIFLHRLKRLRKRLKRK-Ahx-GKPVNHSTRKEEKQRNGT-NH ２ (In the formula, Ac represents that the amino group of the N-terminal amino acid residue is acetylated, Ahx represents ε-aminocaproic acid, and NH ２ indicates that the carboxyl group of the C-terminal amino acid residue is amidated.)

7. A peptide having the following amino acid sequence: Ac-ELKLIFLHRLKRLRKRLKRK-Ahx-SGKPVNHSTRKEEKQRNGT-NH ２ (In the formula, Ac represents that the amino group of the N-terminal amino acid residue is acetylated, Ahx represents ε-aminocaproic acid, and NH ２ indicates that the carboxyl group of the C-terminal amino acid residue is amidated.)

8. A peptide having the following amino acid sequence: Ac-ELKLIFLHRLKRLRKRLKRK-Ahx-ASGKPVNHSTRKEEKQRNGT-NH ２ (In the formula, Ac represents that the amino group of the N-terminal amino acid residue is acetylated, Ahx represents ε-aminocaproic acid, and NH ２ indicates that the carboxyl group of the C-terminal amino acid residue is amidated.)

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