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 any of the amino acid sequences represented by serial numbers 20-22.

Description

Technical Field

[0001] This invention relates to a vaccine composition, and more particularly to a vaccine composition capable of inducing antibodies against IgE in vivo. Background Technology

[0002] IgE is a member of the immunoglobulin family that mediates allergic reactions in type I allergic diseases (such as asthma, allergic rhinitis, urticaria, etc.). The mechanism by which IgE induces type I allergic diseases is briefly described as follows: When IgE binds to the high-affinity IgE receptor (FcεR1) on the surface of mast cells and basophils, and then allergens bind to IgE to form cross-links, mast cells and basophils release chemical mediators, primarily histamine. These released chemical mediators cause vasodilation and increased vascular permeability, resulting in various allergic symptoms.

[0003] As one of the treatments for type I hypersensitivity diseases, omalizumab (trade name Xolair (registered trademark)), an anti-IgE monoclonal antibody, is available on the market. Omalizumab inhibits mast cell activation by specifically binding to and clearing free IgE in the blood, thereby reducing the binding frequency of FcεR1 on the surface of mast cells to IgE.

[0004] Omalizumab has shown some efficacy in treating severe asthma and is one of the effective drugs for treating allergic diseases, but it still has some areas for improvement. For example, omalizumab is a humanized mouse monoclonal antibody, which cannot completely avoid an immune response when used in human patients. Furthermore, antibody drugs are expensive; the cost of omalizumab treatment is reportedly between $15,000 and $44,000 per patient per year. In addition, antibody drugs also have drawbacks such as limitations in administration methods. Against this backdrop, there is an urgent need to develop safer and more cost-effective treatments for allergic diseases.

[0005] To overcome this drawback, alternative solutions such as antigenic peptides that can be administered to organisms to induce anti-IgE antibodies are being actively developed. For example, Patent Document 1 discloses an IgE immunogen construct capable of inducing anti-IgE antibodies. However, the development of IgE immunogens that can efficiently induce anti-IgE antibodies remains a priority.

[0006] Existing technical documents Patent documents Patent Document 1: WO2010067286 Summary of the Invention

[0007] Problems solved by the invention Based on the above background, the purpose of this invention is to provide a novel IgE immunogen that can induce IgE antibodies with extremely high efficiency.

[0008] Problem Solving Methods The inventors of this application have conducted dedicated research on the aforementioned problems and discovered that a complex of a T-cell receptor antigenic peptide with a specific amino acid sequence and a B-cell receptor antigenic peptide with 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 inventors of this application exhibits excellent IgE immunogenicity and can efficiently induce antibodies with high IgE neutralizing activity.

[0009] Based on this discovery, the inventors of this application completed the present invention through further research.

[0010] 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 antigenic peptide and a B-cell receptor antigenic peptide, wherein the B-cell receptor antigenic peptide comprises an amino acid sequence represented by any of serial numbers 20-22. [2] The vaccine composition as described in [1], wherein, The T-cell receptor antigen peptide contains the amino acid sequence represented by sequence number 1. [3] The vaccine composition as described in [1] or [2], wherein, In the complex, binding occurs 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 as described in any one of [1] to [3], wherein, T-cell receptor antigen peptides and B-cell receptor antigen peptides bind through a linker. [5] The vaccine composition as described in any one of [1] to [4] is used to treat or prevent diseases accompanied by excessive secretion of IgE. [6] The vaccine composition as described in [5], wherein, Diseases accompanied by excessive IgE secretion are selected from at least one of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and anaphylactic hypersensitivity.

[0017] [A-1] A method for treating or preventing a disease in a subject with excessive IgE secretion includes administering to the subject a vaccine composition capable of inducing the 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, the B-cell receptor antigen peptide comprising an amino acid sequence represented by any of serial numbers 20-22.

[0018] [A-2] As described in [A-1], wherein, The T-cell receptor antigen peptide contains the amino acid sequence represented by sequence number 1.

[0019] [A-3] The method described in [A-1] or [A-2], wherein, In the complex, binding occurs between the C-terminus of the T-cell receptor antigen peptide and the N-terminus of the B-cell receptor antigen peptide.

[0020] [A-4] The method as described in any one of [A-1] to [A-3], wherein, T-cell receptor antigen peptides and B-cell receptor antigen peptides bind via linkers.

[0021] [A-5] The method as described in any one of [A-1] to [A-4], wherein, Diseases accompanied by excessive IgE secretion are selected from at least one of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and allergic hypersensitivity.

[0022] [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, the vaccine composition being used to treat or prevent diseases accompanied by excessive IgE secretion, the B-cell receptor antigen peptide comprising any of the amino acid sequences represented by serial numbers 20-22.

[0023] [B-2] The vaccine composition as described in [B-1], wherein, The T-cell receptor antigen peptide contains the amino acid sequence represented by sequence number 1.

[0024] [B-3] The vaccine composition as described in [B-1] or [B-2], wherein, In the complex, binding occurs between the C-terminus of the T-cell receptor antigen peptide and the N-terminus of the B-cell receptor antigen peptide.

[0025] [B-4] The vaccine composition as described in any one of [B-1] to [B-3], wherein, T-cell receptor antigen peptides and B-cell receptor antigen peptides bind via linkers.

[0026] [B-5] The vaccine composition as described in any one of [B-1] to [B-4], wherein, Diseases accompanied by excessive IgE secretion are selected from at least one of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and allergic hypersensitivity.

[0027] [C-1] Use of a vaccine composition capable of inducing the production of antibodies against IgE in the manufacture of a medicament for the treatment or prevention of diseases accompanied by excessive IgE secretion, the vaccine composition comprising a complex of a T-cell receptor antigen peptide and a B-cell receptor antigen peptide, the B-cell receptor antigen peptide comprising an amino acid sequence represented by any of serial numbers 20-22.

[0028] [C-2] As described in [C-1], wherein, The T-cell receptor antigen peptide contains the amino acid sequence represented by sequence number 1.

[0029] [C-3] As described in [C-1] or [C-2], wherein, In the complex, binding occurs between the C-terminus of the T-cell receptor antigen peptide and the N-terminus of the B-cell receptor antigen peptide.

[0030] [C-4] The use as described in any one of [C-1] to [C-3], wherein, T-cell receptor antigen peptides and B-cell receptor antigen peptides bind via linkers.

[0031] [C-5] The use as described in any one of [C-1] to [C-4], wherein, Diseases accompanied by excessive IgE secretion are selected from at least one of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and allergic hypersensitivity.

[0032] Invention Effects According to the present invention, the production of anti-IgE antibodies can be induced very efficiently in vivo. Therefore, diseases such as type I hypersensitivity reactions accompanied by excessive IgE secretion can be treated and / or prevented at a low cost and safely. Attached Figure Description

[0033] Figure 1A graph showing the results of antibody titers against synthetic IgE peptides in mouse antiserum measured by ELISA (the mice were administered AJP001 conjugate peptide containing any B-cell epitope sequence from sequence numbers 2–34), and the results of antibody titers against human IgE in mouse antiserum measured by ELISA (the mice were administered AJP001 conjugate peptide containing any B-cell epitope sequence from sequence numbers 16–28). (GMT ± 95% CI (N=4)) Figure 2 A graph showing the results of neutralizing activity assessment (luciferase expression inhibition, mean ± SD (triplicate)) of rat antiserum administered with AJP001 conjugate peptides containing any of the B-cell epitope sequences in sequence numbers 16 and 20–24.

[0034] Figure 3 The figure shows the antibody titers (GMT±95%CI, (N=8)) of each AJP001 conjugate peptide or human IgE in mouse serum when the AJP001 conjugate peptide with sequence number 20 or 21 was administered to humanized IgE / FcεR1 Tg mice.

[0035] Figure 4 A graph showing the concentration of the complex (mean ± SE, (N=7 or 8)) in the serum of humanized IgE / FcεR1 Tg mice after administration of the AJP001 conjugate peptide with B cell epitope sequence number 20 or 21.

[0036] Figure 5 A graph showing the number of nose-scratching behaviors (mean ± SE (N=8), **: p<0.01 vs OVA group (t-test), ##: p<0.01 vs OVA+adjuvant group (t-test)) and the correlation between the number of behaviors and the concentration of the complex (Spearman rank correlation coefficient) when humanized IgE / FcεR1 Tg mice were administered the AJP001 conjugate peptide with sequence number 20 or 21.

[0037] Figure 6 A graph showing the concentrations of OVA-hIgE in the serum of humanized IgE / FcεR1 Tg mice (minimum to maximum (N=4–8)) and the concentrations of eosinophil chemokine (Eotaxin) in NALF (minimum to maximum (N=4–8), *: p<0.05 vs OVA+adjuvant group (t-test)) when the AJP001 conjugate peptide with sequence number 20 or 21 was administered to the mice.

[0038] Figure 7The figure shows the histopathological examination and eosinophilic infiltration in the nasal tissue of humanized IgE / FcεR1 Tg mice after administration of the AJP001 conjugate peptide with B cell epitope sequence number 20 or 21 (mean ± SE (N=4–8), **: P<0.01 vs OVA+adjuvant group (t-test)). Detailed Implementation

[0039] The present invention will now be described in detail.

[0040] 1. Vaccine composition The present invention provides a vaccine composition capable of inducing the production of antibodies against IgE (hereinafter also referred to as "the vaccine composition of the present invention"), 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 any of the amino acid sequences represented by serial numbers 20 to 22.

[0041] The peptides described in this specification are labeled according to peptide notation conventions, with an N-terminus (amino terminus) on the left and a C-terminus (carboxyl terminus) on the right. In the vaccine compositions of this invention, the C-terminus of the peptide complex contained as the active ingredient may optionally be a carboxyl group (-COOH) or a carboxylate group (-COO). - ), amide group (-CONH2) or ester (-COOR).

[0042] Here, R in the ester can be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. C. 1-6 Alkyl groups; such as cyclopentyl, cyclohexyl, etc. (C60) 3-8 Cycloalkyl groups; such as phenyl, α-naphthyl, etc. 6-12 Aryl groups; such as benzyl, phenethyl, etc. phenyl-C 1-2 Alkyl groups; α-naphthylmethyl and other α-naphthyl-C 1-2 Alkyl and other C 7-14 Aryl alkyl groups; neopentyloxymethyl groups, etc.

[0043] When the peptide complex has a carboxyl group (or carboxyl group) other than the C-terminus, the case where the carboxyl group is amidated or esterified is also included in the peptide complex of the present invention. For example, the C-terminal ester described above can be used as the ester in this case.

[0044] Additionally, the peptide complex may also contain amino-protected groups (e.g., formyl, acetyl, etc.) of N-terminal amino acid residues. 1-6 C of alkyl acyl group, etc. 1-6 Complexes protected by acyl groups, complexes in which the amino group of the N-terminal amino acid residue is acetylated, and complexes in which substituents (e.g., -OH, -SH, amino, imidazole, indole, guanidinyl, etc.) on the side chains of intramolecular amino acids are appropriately protected by groups (e.g., formyl, acetyl, etc.).1-6 C of alkyl acyl group, etc. 1-6 A complex protected by acyl groups, etc. 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.

[0045] The peptide complex contained in the vaccine composition of the present invention as an active ingredient contains, in a portion, a B-cell receptor antigenic peptide. Here, a B-cell receptor refers to a receptor expressed on the surface of B cells. Upon stimulation by the antigenic peptide, B cells proliferate and secrete the B-cell receptor as an antibody against the antigenic peptide.

[0046] The B-cell receptor antigen peptide in the vaccine composition of the present invention comprises any of the amino acid sequences represented by serial numbers 20-22. The specific amino acid sequences represented by serial numbers 20-22 are shown below: GKPVNHSTRKEEKQRNGT (Serial Number 20) SGKPVNHSTRKEEKQRNGT (Serial Number 21) ASGKPVNHSTRKEEKQRNGT (Serial Number 22) In one embodiment of the present invention, the B cell receptor antigen peptide may comprise, or be composed of, any of the amino acid sequences represented by sequence numbers 20-22.

[0047] A portion 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 capable of forming a complex with MHC class II, being recognized by the CD3 / TCR complex and CD4, and transmitting signals into CD3-positive cells. MHC class II, for example, includes HLA-DR, HLA-DQ, and HLA-DP in humans, and H-2A or H-2B in mice, which are dimers composed of an α chain and a β chain (e.g., HLA-DR is HLA-DRA as the α chain and HLA-DRB1 as the β chain), preferably HLA-DR, HLA-DQ, or HLA-DP.

[0048] In the vaccine composition of the present invention, there are no particular limitations on the T-cell receptor antigen peptide, as long as it contains an MHC class II molecule that can be presented to antigen-presenting cells to activate helper T cells. In addition to the AJP001 peptide (ELKLIFLHRLKRLRKRLKRK (serial number 1), see WO2016 / 047763) developed by the inventors of this application, other peptides that can be used include UBITh peptides (UBITh (registered trademark) 1: ISITEIKGVIVHRIETILF (serial number 35), UBITh (registered trademark) 2: KKKIITITRIITIITTID (serial number 36), UBITh (registered trademark) 3: ISISEIKGVIVHKIETILF (serial number 37), ISITEIRTVIVTRIETILF (serial number 38), see US Patent No. 9,102,752), tetanus toxoid peptide (TTaa830-843 peptide: QYIKANSKFIGITE (serial number 39)), etc. AJP001 can induce the secretion of IL-β1 and IL-18 through the activation of the NLRP3 inflammasome, and induce the production of TNF-α and IL-6 through the NF-κB pathway, thereby activating the innate immune system. Therefore, it is particularly preferred as a T cell receptor antigen peptide in this invention.

[0049] In the vaccine composition of the present invention, T-cell receptor antigen peptides and B-cell receptor antigen peptides bind to form a complex. This binding can be a connection between the ends of the peptide chains of one and the other, a connection between the amino acid side chains of one peptide and the ends of the other, or an interconnection of the amino acid side chains of both. Preferably, it is a connection between the ends of the peptide chains of one and the other, more preferably, a connection between the C-terminus of one peptide and the N-terminus of the other, and even more preferably, a connection 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 the peptide chains of one and the other are connected, the terminal amino acids can be directly linked by peptide bonds, or they can be linked by a linker (also referred to as a "spacer" in this specification). The linker is not particularly limited, as long as it can connect the T-cell receptor antigen peptide and the B-cell receptor antigen peptide and be taken up into antigen-presenting cells to present the helper T-cell epitopes of the T-cell receptor antigen peptide to free MHC class II molecules. Amino acids other than α-amino acids, such as ε-aminohexanoic acid, β-aminoalanine, γ-aminobutyric acid, 7-aminoheptanoic acid, 12-aminolauric acid, and para-aminobenzoic acid, can be used. Additionally, L-amino acids (e.g., glutamic acid, cysteine, lysine) and their D-amino acids, which are present in natural proteins, can also be used. In a preferred embodiment, the amino acid linker is ε-aminohexanoic acid. Alternatively, peptide linkers consisting of any 2 to 15 amino acids can be used. Examples include G linkers consisting of glycine (Gly) or methylated glycine (MeG), and GS linkers consisting of Gly or MeG and Ser, but are not limited to these. In another embodiment, PEG linkers containing polyethylene glycol (PEG) or polyethylene glycol derivatives can also be used. PEG linkers can also be used that further contain one or more linkers selected from glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg), and lysine (Lys).

[0050] In one embodiment, the complex formed by linking B-cell receptor antigen peptides and T-cell receptor antigen peptides may further include additional amino acids. Such addition of amino acids is permissible as long as the desired effect of the vaccine composition of the present invention is achieved. The added amino acid sequence is not particularly limited, and examples include tags that facilitate the detection and purification of the complex. Examples of tags include Flag tags, histidine tags, c-Myc tags, HA tags, AU1 tags, GST tags, MBP tags, fluorescent protein tags (e.g., GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tags, etc. The position where the amino acid sequence is added is not particularly limited, but is preferably at the N-terminus and / or C-terminus of the complex.

[0051] In another embodiment, besides the label described above, the complex in the vaccine composition of the present invention can also bind to other functional molecules. There are no particular limitations on these functional molecules, as long as they can achieve the desired effect of the vaccine composition of the present invention. Examples of such functional molecules include molecules that inhibit the breakdown of the complex when the vaccine composition of the present invention is administered into a living organism.

[0052] The complex in the vaccine composition of the present invention can be manufactured according to known conventional peptide synthesis schemes, using solid-phase synthesis (Fmoc method and Boc method) or liquid-phase synthesis. When the complex is a B-cell receptor antigen peptide and a T-cell receptor antigen peptide directly linked or linked via amino acids or peptide linkers, the entire complex can be synthesized in one step. Alternatively, the B-cell receptor antigen peptide and the T-cell receptor antigen peptide can be synthesized separately and then directly linked or linked via linkers.

[0053] In one embodiment, the peptide complex in the vaccine composition of the present invention can be coupled to a carrier protein to enhance its immunogenicity. The carrier protein is typically a substance that binds to molecules (haptens) that are not immunogenic due to their small molecular weight to confer immunogenicity, and such substances are known in the art. Examples of carrier proteins include bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), keyhole hemocyanin (KLH), thyroglobulin (TG), diphtheria toxin (CRM197) with a portion of its amino acids replaced to render it non-toxic, and immunoglobulins. The carrier protein can be coupled to the N-terminus or C-terminus of the complex in the vaccine composition of the present invention. As a method of coupling, a cysteine ​​residue can be introduced into the antigenic peptide of the present invention, and coupling can be performed by binding the SH group of the cysteine ​​side chain to the amino group of the carrier protein (MBS method). Alternatively, coupling can be performed by binding between amino groups such as the ε-amino or α-amino group of the lysine residue of the protein (glutaraldehyde method).

[0054] In one embodiment, the vaccine composition of the present invention may further comprise a pharmaceutically acceptable adjuvant that is compatible with the active ingredient. Adjuvants are typically substances that nonspecifically enhance the host's immune response, and most adjuvants are well known in the art. There are no particular limitations on the adjuvants used in the vaccine composition of the present invention, as long as they can nonspecifically enhance the immune response; examples include aluminum salts (Alum), alum, CpG oligodeoxynucleotides, dsRNA, MONTANIDE (a trademark of Seppic), squalene, saponins, etc.

[0055] The vaccine composition of the present invention can be provided as a pharmaceutical composition comprising a pharmaceutically acceptable carrier based on a complex of T-cell receptor antigen peptides and B-cell receptor antigen peptides.

[0056] As a pharmaceutically acceptable carrier, appropriate options can be selected based on the dosage form. 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; flavorings 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; and base waxes, but not limited to these.

[0057] The vaccine composition of this invention can be administered to mammals orally or non-orally. Since the complex of T-cell receptor antigen peptides and B-cell receptor antigen peptides can be degraded in the stomach, non-oral administration is preferred. Examples of formulations suitable for oral administration include liquid formulations, capsules, sachets, tablets, suspensions, emulsions, etc. Examples of formulations suitable for non-oral administration (e.g., subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injectable solutions, which may also contain antioxidants, buffers, antibacterial agents, isotonic agents, etc. Examples of aqueous and non-aqueous sterile suspensions are also included, which may also contain suspending agents, thickeners, saturators, stabilizers, preservatives, etc. This formulation can be sealed in containers such as ampoules or vials according to single-dose or multiple-dose dosages. Furthermore, the active ingredient and pharmaceutically acceptable carrier can be lyophilized and stored in a state where they can be dissolved or suspended in a suitable sterile carrier just before use.

[0058] The content of the active ingredient (i.e., peptide complex) in the vaccine composition is generally 0.001 to 100% by weight of the total composition, preferably 0.05 to 99% by weight, more preferably about 0.1 to 90% by weight, but not limited thereto.

[0059] There are no particular limitations on the recipients of the vaccine composition of the present invention, as long as they are mammals capable of developing a disease with excessive IgE secretion and subsequent disease progression (hereinafter also referred to as "disease with excessive IgE secretion"). Examples of such mammals include rodents such as mice, pets such as dogs and cats, livestock such as pigs, horses and cattle, humans, monkeys, orangutans and chimpanzees, and humans are particularly preferred.

[0060] The dosage of the vaccine composition of the present invention varies depending on the target population, method of administration, and form of administration. Generally, for an adult, the dosage ranges from 1 μg to 300,000 μg per dose, preferably from 20 μg to 30,000 μg. The complex of T-cell receptor antigen peptide and B-cell receptor antigen peptide, which is the active ingredient, is usually administered 2 to 3 times over 4 to 12 weeks. An additional dose can be administered each time the antibody titer decreases.

[0061] Diseases that can be treated or prevented by the vaccine composition of the present invention are the aforementioned diseases accompanied by excessive IgE secretion. Examples of such diseases include, but are not limited to, asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and allergic hypersensitivity reactions.

[0062] In one embodiment, the vaccine composition of the present invention can be used in combination with existing therapeutic agents for diseases accompanied by excessive IgE secretion. For example, in the treatment of allergic diseases, treatment can be initiated with a rapid-acting antibody drug (e.g., omalizumab) 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 a balance between patient quality of life and treatment costs. Furthermore, the vaccine composition of the present invention can also be used simultaneously with existing therapeutic agents.

[0063] It should be noted that the term "treatment" in this instruction manual includes not only the cure of the disease, but also the relief of the disease and the improvement of the severity of the disease.

[0064] In addition to preventing the onset of disease, the term "prevention" in this instruction manual also includes delaying the onset of disease. Furthermore, the term "prevention" in this instruction manual also includes preventing or delaying the recurrence of disease after treatment.

[0065] Additionally, the term "vaccine composition" in this specification may be replaced with "pharmaceutical composition" or "pharmaceutical".

[0066] 2. Methods for treating or preventing diseases accompanied by excessive IgE secretion. The present invention also provides a method for treating or preventing diseases with excessive IgE secretion (hereinafter also referred to as the "method of the present invention"), which includes the step of administering the vaccine composition of the present invention to a subject who has or may have a disease with excessive IgE secretion.

[0067] The treatment or prevention targets in the method of the present invention, the administration conditions of the vaccine composition of the present invention, etc. are the same as those described in "1. Vaccine Composition".

[0068] It should be noted that, in one embodiment, the present invention provides a method for inducing the production of antibodies against IgE in a subject, comprising the step of administering the vaccine composition of the present invention to the subject. The subject, administration conditions of the vaccine composition, etc., in this method are the same as those described in "1. Vaccine Composition".

[0069] The invention is illustrated in more detail in the following embodiments, but the invention is not limited to these examples.

[0070] Example Peptide synthesis (Fmoc method) Following the method described in Lecture 16, Organic Compound Synthesis IV, of Experimental Chemistry, 5th Edition, a protective peptide resin was synthesized using an automated solid-phase synthesizer via the Fmoc method. Trifluoroacetic acid (TFA) and a scavenging agent (a mixture of thioanisole, 2,2'-(ethylenedioxy)diethylthiol, m-cresol, triisopropylsilane, and water) were added to the obtained protective peptide resin to cleave the peptide from the resin and simultaneously deprotect it, yielding a crude peptide. The crude peptide was purified using a reversed-phase HPLC column with gradient elution in a 0.1% TFA-H₂O / CH₃CN system. The fraction containing the target analyte was collected and lyophilized to obtain the target peptide.

[0071] HPLC analysis method for peptides The purity of the synthetic peptides was determined using an HPLC apparatus under the following analytical conditions.

[0072] HPLC model: Shimadzu LCLC-20ADXR Measurement wavelength: 220 nm Flow rate: 0.31 mL / min Column: Inertsil ODS-3, 2.1m × 250mm, 5μm Column temperature: room temperature Mobile phase A: 0.1% trifluoroacetic acid aqueous solution Mobile phase B: Acetonitrile Gradient conditions: The concentration of mobile phase B increases linearly from 5% to 80% over 30 minutes (5→80% mobile phase B, over 30 minutes). Mass spectrometry analysis of peptides The quality of the synthetic peptides was determined using MALDI-TOF-MS under the following analytical conditions.

[0073] MALDI-TOF-MS model: Bruker autoflex speed Matrix: 2,5-Dihydroxybenzoic acid Solution: A mixture of 0.1% trifluoroacetic acid aqueous solution and acetonitrile [Example 1] Immunogenicity evaluation test in rats using AJP001 conjugate peptide (human IgE) The human IgE epitope peptides (serial numbers 2-34) shown in Table 2 were selected as B-cell antigens, and the T-cell antigen AJP001 (Table 1, serial number 1) and ε-aminocaproic acid (also known as "Ahx") were used as spacers to prepare a coupled complex (AJP001 coupled peptide) (commissioned to Toray Research Center, Inc. or Peptide Research Institute, Inc.). It should be noted that in this specification, the AJP001 coupled peptide with the amino acid sequence represented by serial number "X" on the B-cell epitope is referred to as "AJP001 coupled peptide (serial number X)", etc.

[0074] Table 1 Table 2 AJP001-coupled peptides containing any B-cell epitope sequence from sequence numbers 2 to 34 were dissolved in physiological saline and mixed with 2% Alhydrogel (Invivogen, 0.3 mg / body) and K3 Et-Free (Genedesign, 0.1 mg / body). The mixture was administered subcutaneously to JCL:Wistar rats (female, 7 weeks old, N=4) at 0.5 mg / body doses, spaced two weeks apart. Blood samples were collected before administration and 6 weeks after the first administration, and antibody titers against each epitope sequence and human IgE were determined by ELISA. Specifically, for 96-well plates containing epitope peptides dissolved to 10 μg / mL in carbonated buffer, the plates were blocked with 5% skim milk / PBS, and then serially diluted serum (using 5% skim milk / PBS) was added. The plates were incubated 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 plates were shaken at room temperature for 3 hours. After washing the wells with PBS-T, TMB solution (SIGMA) was added, and the mixture was incubated in the dark for 30 minutes. The reaction was then terminated by adding 0.1 M H₂SO₄ (Kanto Chemical). The absorbance at 450 nm was measured using a plate reader. The serum dilution factor at half the maximum absorbance (OD=1.75) was defined as the antibody titer, and the geometric mean titer (GMT) was calculated from individual values. The figure shows the 95% confidence interval (95% CI) of the geometric mean titer (GMT). Antibody titers against human IgE were measured using full-length human IgE protein (abcam) as a solid-phase antigen, and the same ELISA method was used as described above.

[0075] Figure 1The antibody titers against each AJP001 conjugate peptide and human IgE were displayed. The results showed that human IgE antibody titers were significantly increased in rat antiserum administered with AJP001 conjugate peptides containing B-cell epitope sequences 17–27.

[0076] [Example 2] Evaluation of the neutralizing activity of rat antiserum against AJP001-coupled peptide (human IgE) The neutralizing activity of anti-human IgE antibodies produced by the AJP001 conjugate peptide was evaluated using serum samples collected 6 weeks after the initial administration of the drug in Example 1. A stable expression cell line (RBL-2H3 / FCER1A+NFAT NLuc cells) was established by introducing the human FcεRIα and NFAT-RE-luciferase genes into RBL-2H3 cells. These cells bind to FcεRIα upon IgE supplementation, and then NFAT, the reporter gene (luciferase), is activated and expressed by stimulation with the anti-human IgE antibody. Inhibition of luciferase expression during human IgE stimulation was used as an indicator for evaluation. Specifically, for sequences 16, 20-24, pooled serum (400 μL / individual, N=4) was prepared, inactivated (56°C, reaction for 30 min), and then an equal volume of ammonium sulfate (Fujifilm and Kodenpaku) was added. The mixture was inverted and reacted at room temperature for 30 min. Subsequently, the precipitate was centrifuged at 4°C and 3000×g for 20 minutes, dissolved in PBS buffer, and serum IgG antibodies (including anti-IgE antibodies) were purified using Protein G HP spin trap (Cytiva), 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 culture medium (DMEM containing 1% P / S, 400 μg / mL G418, 500 μg / mL hygromycin B, and 10% FBS) and incubated at 37°C for 2 hours. Then, 50 μL / well of cells (1.0 × 10^5 cells / 50 μL / well) were added to each well of a 96-well ThermoFisher plate and incubated at 37°C for 24 hours. Note that untreated wells and control wells containing only human IgE were included. After 24 hours, the plates were washed with maintenance medium, and anti-human IgE antibody (final concentration 1 μg / mL, AQI) was added and incubated at 37°C for 4 hours. Afterward, NanoGlo luciferase detection substrate was added, and the reaction was allowed to proceed for 3 minutes. The luminescence value was then measured using a luminescence plate reader. Based on the luminescence values ​​of the untreated wells (A), control wells (B), and wells (C) containing purified IgG from rat antiserum injected with each AJP001 conjugate peptide, the percentage (%) of the luminescence value relative to the control wells was calculated using the following formula.

[0077] 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. Neutralizing activity was expressed in steps based on the concentration (μg / mL) of IC50 (+: 10^4, ++: 10^3, +++: 10^2, NC if not calculable).

[0078] The percentage of luminescence relative to the control aperture (%) = 100 × [(CA) / (BA)] Figure 2 Neutralizing activity against human IgE was demonstrated. The results showed that neutralizing activity (inhibition of luciferase expression) was confirmed in rat antiserum administered with the AJP001 conjugate peptide containing B-cell epitope sequences numbered 20, 21, and 22.

[0079] [Example 3] Using humanized IgE / FcεR1 Tg mice in an OVA-induced allergic rhinitis model, AJP001 was used to treat the condition. Pharmacodynamic evaluation test of conjugated peptide (human IgE) The AJP001 conjugate peptide, containing B-cell epitope sequences of sequence number 20 or 21, was dissolved in physiological saline and mixed with 2% Alhydrogel (Invivogen, 0.3 mg / body) and K3 Et-Free (Genedesign, 0.1 mg / body). It was then subcutaneously administered to humanized IgE / FcεR1 Tg mice (female, 10 weeks old, N=8 / group) at 0.5 mg / body, at two-week intervals, for a total of three doses (days 0, 14, and 28). One hour before OVA administration on days 56–66, an antihistamine (Bilastine, Dapeng Pharmaceutical Co., Ltd.) was administered orally at 50 mg / kg as a positive control. The OVA + adjuvant group served as the control group for the sequence number 20 or 21 administration group, and the OVA group served as the control group for the Bilastine administration group. The disease model was established by administering OVA (containing alum) at 100 μg / body (intraperitoneal) on day 35, 50 μg / body (subcutaneous) on day 49, and 50 μg OVA intranasally on days 56–66. Under these experimental conditions, serum was collected on days 0, 14, 28, 35, 49, and 67, and antibody titers against each AJP001 conjugate peptide and human IgE, serum concentrations of human IgE / anti-human IgE antibody complexes, and serum concentrations of OVA-specific human IgE were determined by ELISA. Furthermore, video recordings were taken 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 wash fluid (NALF) was collected, and the concentration of eosinophil chemokine (Eotaxin) in NALF was determined by ELISA. In addition, after collecting nasal tissue, Luna-stained specimens were prepared, and the number of eosinophil infiltrations was determined. Antibody titers were determined using the same procedures as in Example 1. Serum OVA-specific human IgE concentrations were measured using the Human Ovalbumin Specific IgE ELISA Kit (Finetest), and NALF Eotaxin concentrations were measured using the Mouse CCL11 / Eotaxin immunoassay kit (R&D).

[0080] Figure 3 The antibody titers against each AJP001 conjugate peptide and human IgE are shown. Figure 4 The concentration of the complex in the serum was shown. Figure 5 A graph showing the correlation between the number of nose-scratching behaviors and the concentration of the complex was displayed. Figure 6 The serum OVA-hIgE concentration and the Eotaxin concentration in NALF were displayed. Figure 7 It shows the histopathological examination and the degree of eosinophil infiltration.

[0081] The results showed that in the groups administered sequence numbers 20 or 21, antibody titers increased over time, significantly inhibiting the frequency of nose scratching and eosinophil infiltration in the nasal tissue. Furthermore, the concentrations of Eotaxin in NALF and serum OVA-specific human IgE also showed a downward trend. These results suggest that the antibodies induced in the groups administered sequence numbers 20 or 21 can suppress rhinitis symptoms by binding to human IgE and inhibiting IgE / FcεR1 binding. On the other hand, the antihistamine (bilastine) showed equivalent efficacy in reducing the frequency of nose scratching as in the groups administered sequence numbers 20 or 21, with other parameters remaining unchanged. This indicates that the IgE antibody-induced peptide and the antihistamine inhibit rhinitis symptoms through different mechanisms of action.

[0082] Industrial availability According to the present invention, peptide vaccines for the treatment and / or prevention of diseases such as type I allergic diseases accompanied by excessive IgE secretion can be manufactured at low cost, which is of great use in the pharmaceutical field.

[0083] This application is based on Japanese Patent Application No. 2023-191836 (application date: November 9, 2023), the entire contents of which are included in this specification.

Claims

1. A vaccine composition capable of inducing the production of antibodies against IgE, comprising a complex of a T-cell receptor antigenic peptide and a B-cell receptor antigenic peptide, wherein the B-cell receptor antigenic peptide comprises an amino acid sequence represented by any one of serial numbers 20-22.

2. The vaccine composition of claim 1, wherein, The T-cell receptor antigen peptide contains the amino acid sequence represented by sequence number 1.

3. The vaccine composition according to claim 1 or 2, wherein, In the complex, binding occurs 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, T-cell receptor antigen peptides and B-cell receptor antigen peptides bind via linkers.

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

6. The vaccine composition of claim 5, wherein, Diseases accompanied by excessive IgE secretion are selected from at least one of asthma, allergic asthma, allergic rhinitis, conjunctivitis, eczema, urticaria, atopic dermatitis, and allergic hypersensitivity.

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