Vaccine composition for inducing Anti-ige antibody

Abstract

To provide a polypeptide capable of inducing an anti-IgE antibody extremely efficiently in vivo.SOLUTION: The present invention provides a vaccine composition capable of inducing production of an antibody against IgE, the vaccine composition containing a complex of a T-cell receptor antigen peptide and a B-cell receptor antigen peptide, wherein the B-cell receptor antigen peptide includes an amino acid sequence represented by any one of SEQ ID NOs: 20-22.SELECTED DRAWING: None

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 vivo.

Background Art

[0002] IgE is a type of immunoglobulin family that mediates allergic responses in type I allergic diseases (such as asthma, allergic rhinitis, urticaria, etc.). The mechanism by which IgE causes type I allergic diseases is briefly as follows: When IgE binds to the high-affinity IgE receptor (FcεR1) present on the cell surface of mast cells and basophils, and further, when an allergen binds to the IgE and forms a crosslink, mast cells and basophils release chemical mediators such as histamine. The released chemical mediators enhance blood vessel dilation and vascular permeability. As a result, various allergic symptoms occur.

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

[0004] Omalizumab has a certain effect on severe asthma and is one of the effective therapeutic agents for allergic diseases, but still has some points for improvement. For example, omalizumab is a humanized mouse monoclonal antibody and cannot completely avoid immunological reactions when used in human patients. In addition, antibody drugs are expensive, and the cost associated with treatment using omalizumab is said to be $15,000 to $44,000 per patient per year. Furthermore, antibody drugs also have demerits such as limited administration methods. Against this background, the development of a safer and lower-cost treatment method for allergic diseases is strongly demanded.

[0005] In order to overcome such demerits, development of alternative means such as antigenic peptides that can induce anti-IgE antibodies by administration to a living body has been actively pursued. For example, Patent Document 1 discloses an IgE immunogen construct that can induce anti-IgE antibodies. However, development of an IgE immunogen that can efficiently induce anti-IgE antibodies has always been demanded.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Based on the above-described background, an object of the present invention is to provide a novel IgE immunogen that can extremely efficiently induce anti-IgE antibodies.

Means for Solving the Problems

[0008] As a result of intensive studies on the above 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 in the Fc region of IgE can extremely efficiently induce anti-IgE neutralizing antibodies in vivo. The complex found by the present inventors has excellent IgE immunogenicity and can efficiently induce an antibody having high IgE neutralizing activity. Based on such findings, the present inventors have further advanced research and 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 according to [1], wherein the T cell receptor antigen peptide comprises the amino acid sequence represented by SEQ ID NO: 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 bound via a linker. [5] The vaccine composition according to any one of [1] to [4] for the treatment or prevention of a disease accompanied by excessive secretion 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 the treatment or prevention of a disease accompanied by excessive secretion of IgE in a subject, comprising administering to the subject 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. [A-2] The method according to [A-1], wherein the T cell receptor antigen peptide comprises the amino acid sequence represented by SEQ ID NO: 1. [A-3] The method according to [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 a T cell receptor antigen peptide and a 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 an antibody 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 of SEQ ID NOs: 20 to 22. [B-2] The vaccine composition for use according to [B-1], wherein the T cell receptor antigen peptide comprises an amino acid sequence represented by SEQ ID NO: 1. [B-3] The 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 a T cell receptor antigen peptide and a 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 comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide for inducing the production of an antibody against IgE in the manufacture of a medicament for treating or preventing a disease associated with over-secretion of IgE, wherein the B cell receptor antigen peptide comprises an amino acid sequence represented by any of SEQ ID NOs: 20 to 22. [C-2] The use according to [C-1], wherein the T cell receptor antigen peptide comprises the amino acid sequence represented by SEQ ID NO: 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 bound via a linker. [C-5] The use according to any one of [C-1] to [C-4], wherein the disease associated with over-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, the production of anti-IgE antibodies can be induced very efficiently in vivo. Therefore, inexpensive and safe treatment and / or prevention of diseases associated with over-secretion of IgE such as type I allergic diseases become possible. [Brief Description of the Drawings]

[0011]

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[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, which comprises 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 of SEQ ID NOs: 20 to 22 (hereinafter sometimes referred to as "the vaccine composition of the present invention").

[0014] The peptides described in this specification have the N-terminal (amino terminus) at the left end and the C-terminal (carboxyl terminus) at the right end according to the convention of peptide labeling. The peptide complex contained as an active ingredient in the vaccine composition of the present invention has a C-terminal carboxyl group (-COOH), carboxylate (-COO - )), amide (-CONH2) or ester (-COOR).

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

[0016] When the peptide complex has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the peptide complex of the present invention. As the ester in this case, for example, the C-terminal ester described above is used.

[0017] Furthermore, in the peptide complex, those in which the amino group of the N-terminal amino acid residue is protected by a protecting group (for example, C such as formyl group, acetyl group, etc. 1-6 alkanoyl, etc., C 1-6 acyl group, etc.), those in which the amino group of the N-terminal amino acid residue is acetylated, substituents on the side chain of the amino acid in the molecule (for example, -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (for example, C such as formyl group, acetyl group, etc. 1-6 alkanoyl group, etc., C 1-6 acyl group, etc.) are also included. In one embodiment, the amino group of the N-terminal amino acid residue of the peptide complex is acetylated and / or the carboxyl group of the C-terminus is amidated.

[0018] The peptide complex contained as an active ingredient in the vaccine composition of the present invention contains a B cell receptor antigen peptide in 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 contains an amino acid sequence represented by any of SEQ ID NOs: 20 to 22. The amino acid sequences represented by SEQ ID NOs: 20 to 22 are specifically as follows:

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

[0021] In one aspect of the present invention, the B cell receptor antigen peptide may contain or consist of an amino acid sequence represented by any of SEQ ID NOs: 20 to 22.

[0022] A 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 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, and is a dimer consisting of an α chain and a β chain (for example, HLA-DR is HLA-DRA as the α chain and HLA-DRB1 as the β chain), and 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 MHC class II molecules of antigen-presenting cells and activate helper T cells. For example, in addition to the AJP001 peptide (ELKLIFLHRLKRLRKRLKRK (SEQ ID NO: 1), for details, see WO2016 / 047763) developed by the present inventors, 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), for details, see US patent no. 9,102,752), Tetanous toxisoid peptide (TTaa830-843 peptide: QYIKANSKFIGITE (SEQ ID NO: 39)), etc. can be used. AJP001 can also activate the innate immune system by inducing the secretion of IL-β1 and IL-18 through the activation of the NLRP3 inflammasome and inducing the production of TNF-α and IL-6 through the NF-κB pathway, and thus is particularly preferable as the T cell receptor antigen peptide in the present invention.

[0024] In the vaccine composition of the present invention, the T cell receptor antigen peptide and the B cell receptor antigen peptide bind to form a complex. The binding may be a linkage between the ends of one peptide chain and the ends of the other peptide chain, or a binding between the amino acid side chain of one peptide and the end of the other peptide chain, or a binding between the amino acid side chains of both, but preferably it is a linkage between the ends of one peptide chain and the ends of the other peptide chain, more preferably a linkage between the C-terminus of one peptide chain and the N-terminus of the other peptide chain, and even more preferably a linkage between the C-terminus of the T cell receptor antigen peptide and the N-terminus of the B cell receptor antigen peptide. When the ends of one peptide chain and the ends of the other peptide chain are linked, the terminal amino acids may be directly linked by a peptide bond, or may be linked via a linker (also referred to as a "spacer" in this 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, can be taken up into antigen-presenting cells, and can release the helper T cell epitope in the T cell receptor antigen peptide and present it on MHC class II molecules. For example, amino acids other than α-amino acids such as ε-aminocaproic acid, β-alanine, γ-aminobutyric acid, 7-aminoheptanoic acid, 12-aminolauric acid, p-aminobenzoic acid can be used. Also, L-amino acids (e.g., glutamic acid, cysteine, 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 also be used. For example, a G linker which is a peptide linker consisting of glycine (Gly) or methylated glycine (MeG), a GS linker which is a peptide linker consisting of Gly or MeG and Ser, etc. can be mentioned, but it is not limited thereto. In another embodiment, a PEG linker containing polyethylene glycol (PEG) or a derivative of polyethylene glycol can also 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 aspect, the complex in which a B cell receptor antigen peptide and a T cell receptor antigen peptide are linked may further contain additional amino acids. Such addition of amino acids is acceptable as long as the desired effects of the vaccine composition of the present invention can be obtained. The added amino acid sequence is not particularly limited, and examples thereof include tags for facilitating detection and 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 tags (e.g., GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, and the like. The position where the amino acid sequence is added is not particularly limited, but is preferably the N-terminus and / or C-terminus of the complex. Also, in another aspect, the complex in the vaccine composition of the present invention may be bound to other functional molecules other than the tags described above. Such functional molecules are not particularly limited as long as the vaccine composition of the present invention exhibits the desired effects. Examples of such functional molecules include, but are not limited to, molecules having a function of suppressing the degradation of the complex when the vaccine composition of the present invention is administered in vivo.

[0026] The complex in the vaccine composition of the present invention can be produced by a solid-phase synthesis method (Fmoc method and Boc method) or a liquid-phase synthesis method according to known general peptide synthesis protocols. When the complex is one in which a B cell receptor antigen peptide and a T cell receptor antigen peptide are directly linked or linked via an amino acid or a peptide linker, the entire complex 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 directly linked or linked via a linker.

[0027] In one aspect, 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) having no immunogenicity due to its small molecular weight and confers immunogenicity, and some 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) in which some amino acids of diphtheria toxin are replaced to make it non-toxic, immunoglobulins, 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 of conjugation, a cysteine residue can be introduced into the antigen peptide of the present invention, and it can be conjugated by binding to the amino group of the carrier protein via the SH group which is the side chain of the cysteine (MBS method). Also, it can be conjugated by binding amino groups such as the ε-amino group or α-amino group of the lysine residue of the protein (glutaraldehyde method).

[0028] In one aspect, the vaccine composition of the present invention may further contain an adjuvant that is pharmaceutically acceptable and compatible with the active ingredient. An adjuvant is generally a substance that nonspecifically enhances the immune response of the 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 nonspecifically enhance the immune response, and examples thereof include aluminum salts (Alum), alum, CpG oligodeoxynucleotides, dsRNA, Montanide (trade name of Seppic), squalane, saponin, and the like.

[0029] The vaccine composition of the present invention can be provided as a pharmaceutical composition containing a pharmaceutically acceptable carrier in addition to the complex of the T cell receptor antigen peptide and the B cell receptor antigen peptide.

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

[0031] The vaccine composition of the present invention can be administered to mammals orally or parenterally. Since the complex of the T cell receptor antigen peptide and the B cell receptor antigen peptide can be decomposed in the stomach, it is preferably administered parenterally. Formulations suitable for oral administration include solutions, capsules, sachets, tablets, suspensions, emulsions, etc. Formulations suitable for parenteral administration (for example, 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, tonicity agents, etc. Further, aqueous and non-aqueous sterile suspension solutions are included, which may contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, preservatives, etc. The said formulations can be enclosed in containers in unit doses or multiple doses like ampoules and vials. Further, the active ingredient and the pharmaceutically acceptable carrier can be lyophilized and stored in a state where they can be dissolved or suspended in an appropriate sterile vehicle immediately before use.

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

[0033] The administration target of the vaccine composition of the present invention is not particularly limited as long as it is a mammal that may suffer from a disease whose condition may deteriorate with the excessive secretion of IgE (hereinafter sometimes referred to as "disease accompanied by excessive secretion of IgE"). Such mammals include, for example, rodents such as mice, pets such as dogs and cats, livestock such as pigs, horses, and cows, humans, and primates such as monkeys, orangutans, and chimpanzees, and humans are particularly preferred.

[0034] The dosage of the vaccine composition of the present invention varies depending on the administration target, administration method, administration form, etc., but usually, per adult, a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide as an active ingredient is in the range of 1 μg to 300,000 μg per administration, preferably in the range of 20 μg to 30,000 μg, and is usually administered 2 to 3 times over 4 to 12 weeks. When the antibody titer decreases, it can be additionally administered once each time.

[0035] The diseases that can be treated or prevented by the vaccine composition of the present invention are the above-mentioned diseases accompanied by excessive secretion of IgE. Such diseases include, for example, asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and anaphylactic hypersensitivity, but are not limited thereto.

[0036] In one aspect, the vaccine composition of the present invention may be used in combination with an existing therapeutic agent for diseases accompanied by excessive secretion of IgE. For example, in the treatment of allergic diseases, in the active stage of the disease, treatment is started with an antibody drug (e.g., omalizumab, etc.) with high immediate efficacy, and in the remission stage, administration of the vaccine composition of the present invention is started, so that both the patient's QOL and treatment cost can be balanced. Also, the vaccine composition of the present invention and an existing therapeutic drug can be used simultaneously.

[0037] Note that the "treatment" of a disease in this specification may include not only the cure of the disease, but also the remission of the disease and the improvement of the degree of the disease.

[0038] In addition, the "prevention" of a disease in this specification includes, in addition to preventing the onset of the disease, delaying the onset of the disease. In addition, the "prevention" of a disease in this specification 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" in this specification can also be rephrased as "pharmaceutical composition" or "agent".

[0040] 2. Method for treating or preventing diseases accompanied by excessive secretion of IgE The present invention also provides a method for treating or preventing a disease accompanied by excessive secretion of IgE (hereinafter sometimes referred to as "the method of the present invention"), which includes administering the vaccine composition of the present invention to a subject suffering from or likely to suffer from a disease accompanied by excessive secretion of IgE.

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

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

[0043] The present invention will be further specifically described in the following examples, but the present invention is not limited by these examples.

Examples

[0044] Synthesis of peptide (Fmoc method) According to the method described in the 5th Edition of Experimental Chemistry Course 16: Synthesis of Organic Compounds IV, etc., a protected peptide resin was synthesized using an automatic solid-phase synthesizer by the Fmoc method. Trifluoroacetic acid (TFA) and a scavenger (a mixture of thioanisole, 2,2'-(ethylenedioxy)diethanethiol, m-cresol, triisopropylsilane, and water, etc.) were added to the obtained protected peptide resin, and it was cleaved from the resin and deprotected to obtain a crude peptide. This crude peptide was subjected to gradient elution in a 0.1% TFA-H20 / CH3CN system using a reverse-phase HPLC column for purification. The fractions containing the target product were collected and freeze-dried to obtain the target peptide.

[0045] HPLC analysis method of peptide The purity of the synthesized peptide was measured by an HPLC apparatus under the following analysis conditions. HPLC model: Shimadzu LCLC-20ADXR Measurement wavelength: 220 nm Flow rate: 0.31 mL per minute Column: Inertsil ODS-3, 2.1m×250mm, 5 micron Column temperature: Room temperature Mobile phase A: 0.1% aqueous trifluoroacetic acid solution Mobile phase B: Acetonitrile Gradient condition: The concentration of mobile phase B is linearly gradiented from 5% to 80% in 30 minutes. (5→80% buffer B in 30min)

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

[0047] [Example 1] Rat immunogenicity evaluation test using AJP001 conjugated peptide (human IgE) The epitope peptides of human IgE shown in Table 2 (SEQ ID NOs: 2 to 34) were selected as B cell antigens, and a conjugate (AJP001 conjugate peptide) was prepared by conjugating T cell antigen AJP001 (Table 1, SEQ ID NO: 1) and ε-aminocaproic acid (sometimes referred to as "Ahx") as a spacer (the 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)", etc.

[0048]

Table 1

[0049]

Table 2

[0050] The AJP001 conjugate peptide having a B cell epitope sequence of any one of SEQ ID NOs: 2 to 34 was dissolved in physiological saline, mixed with 2% Alhydrogel (invivogen, 0.3 mg / body) and K3 Et-Free (GeneDesign, 0.1 mg / body), and subcutaneously administered to JCL: Wistar rats (female, 7 weeks old, N = 4) at 0.5 mg / body three times at two-week intervals. Blood was collected before administration and 6 weeks after the first administration, and the antibody titers against each epitope sequence and human IgE were measured by ELISA. Specifically, after blocking a 96-well plate immobilized with an epitope peptide dissolved in carbonate buffer at 10 μg / mL with 5% skim milk / PBS, serum serially diluted with 5% skim milk / PBS was added and left standing 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 the mixture was shaken at room temperature for 3 hours. After washing the wells with PBS-T, TMB solution (SIGMA) was added and left standing in the dark for 30 minutes, 0.1 M 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 reached 1 / 2 of the maximum value (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% 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 immobilized antigen.

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

[0052] [Example 2] Neutralization activity evaluation using rat antiserum of AJP001 conjugated peptide (human IgE) Using the sera at 6 weeks after the first administration in Example 1, the neutralizing activity of the anti-human IgE antibody produced by the AJP001 conjugate peptide was evaluated. A stable cell line (RBL-2H3 / FCER1A+NFAT NLuc cells) established by introducing the human FcεRIα and NFAT-RE-Luciferase genes into RBL-2H3 was used. When human IgE is added to this cell, it binds to FcεRIα, and then NFAT is activated by stimulation with an anti-human IgE antibody, and the reporter gene (Luciferase) is expressed. The inhibition of Luciferase expression in human IgE stimulation was used as an index for evaluation. Specifically, for SEQ ID NOs: 16 and 20 to 24, pooled sera (400 μL / individual, N = 4) were prepared. After inactivation treatment (reaction at 56°C for 30 minutes), an equal amount of ammonium sulfate (FUJIFILM Wako Pure Chemical Corporation) was added, and the mixture was reacted at room temperature for 30 minutes with inversion and mixing. Then, it was centrifuged at 4°C, 3000×g for 20 minutes, the precipitate was dissolved in PBS, and IgG antibodies (including anti-IgE antibodies) in the sera were purified using Protein G HP spin trap (Cytiva), Ab buffer kit (Cytiva), and Amicon ultra-0.5 centrifugal filter devices (100K) (Millipore). Next, this purified IgG (final concentration 1.2 to 500 μg / mL) and human IgE (final concentration 0.6 μg / mL) were added to a culture medium (DMEM containing 1% P / S, 400 μg / mL G418, 500 μg / mL hygromycin B, 10% FBS), reacted at 37°C for 2 hours, and then added to the cells (1.0 × 10^5 cells / 50 μL / well) seeded in a 96-Well white plate (ThermoFisher) at 50 μL / well, and reacted at 37°C for 24 hours. In addition, untreated wells and control wells with only human IgE added were set. After 24 hours, the cells were washed with maintenance medium, an anti-human IgE antibody (final concentration 1 μg / mL, AQI) was added, and the mixture was reacted at 37°C for 4 hours. Then, NanoGlo Luciferase Assay substrate was added, reacted for 3 minutes, and the luminescence value was measured with a luminometer plate reader.The ratio (%) of the luminescence value to that of the control well was calculated from the luminescence values of each well of untreated well (A), control well (B), and well (C) of purified IgG derived from rat antiserum administered with each AJP001 conjugate peptide using the following formula.

[0053] Based on the added concentration of the purified IgG, 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 stepwise from the concentration (μg / mL) of IC50 (+: 10^4 digits, ++: 10^3 digits, +++: 10^2 digits, and when calculation was not possible, denoted as NC).

[0054] Ratio (%) of luminescence value to that of the control well = 100 × [(C - A) / (B - A)]

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

[0056] [Example 3] Pharmacodynamic evaluation test on OVA-induced allergic rhinitis model using Humanized IgE / FcεR1 Tg mice of AJP001 conjugated peptide (human IgE) The 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 (GeneDesign, 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, three times at two-week intervals (Day 0, 14, 28). The positive control substance, an antihistamine drug (Bilastine, Taisho Pharmaceutical Co., Ltd.), was orally administered at 50 mg / kg one hour before OVA administration from Day 56 to 66. Also, an OVA + adjuvant group was set as the control group for the SEQ ID NO: 20 or 21 administration group, and an OVA group was set as the control group for the Bilastine administration group. The disease model was prepared by administering OVA (containing Alum) intraperitoneally at 100 μg / body on Day 35 and subcutaneously at 50 μg / body on Day 49, and then administering 50 μg of OVA intranasally from Day 56 to 66. Under the conditions of this test, sera (Day 0, 14, 28, 35, 49, and 67) were collected, and the antibody titers against each AJP001 conjugate peptide and human IgE, the complex concentration of human IgE / anti-human IgE antibody in the serum, and the OVA-specific human IgE concentration in the serum were measured by ELISA. Also, video recording was performed one hour after intranasal administration on Day 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, after collecting nasal tissues, Luna-stained specimens were prepared, and the number of eosinophil infiltrations was measured. The measurement of antibody titers was performed in the same procedure as in Example 1, the OVA-specific human IgE concentration in the serum was measured using a Human Ovalbumin Specific IgE ELISA Kit (Finetest), and the Eotaxin concentration in the NALF was measured using a Mouse CCL11 / Eotaxin immunoassay kit (R&D).

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

[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 scratching and eosinophil infiltration in the nasal tissue were significantly suppressed. In addition, a tendency towards lower values was also shown in the Eotaxin concentration in NALF and the OVA-specific human IgE concentration in serum. From these results, it was suggested that the antibody induced in the group administered with SEQ ID NO: 20 or 21 binds to human IgE and inhibits IgE / FcεR1 binding, thereby suppressing rhinitis symptoms. On the other hand, the antihistamine drug (bilastine) showed the same efficacy as the group administered with SEQ ID NO: 20 or 21 in terms of the number of nose scratching, but the other parameters did not change. From this, it was suggested that this IgE antibody-inducing peptide and the antihistamine drug suppress rhinitis symptoms by different mechanisms of action.

Industrial Applicability

[0059] According to the present invention, a peptide vaccine for the treatment and / or prevention of diseases accompanied by excessive secretion of IgE such as type I allergic diseases can be produced at low cost, and thus it is extremely useful in the field of pharmaceutical production.

[0060] This application is based on Japanese Patent Application No. 2023-191836 (filing date: November 9, 2023), the content of which is entirely incorporated 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 comprises an amino acid sequence represented by any one of SEQ ID NOs: 20 to 22.

2. The vaccine composition according to claim 1, wherein the T cell receptor antigen peptide comprises an amino acid sequence represented by SEQ ID NO:

1.

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

5. The vaccine composition according to claim 1 or 2 for the treatment or prevention of diseases accompanied by excessive secretion of IgE.

6. The vaccine composition according to claim 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.

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