Vaccine composition for inducing Anti-il-23 antibody

Abstract

The present invention provides a vaccine composition which contains a complex of a T-cell receptor antigen peptide and a B-cell receptor antigen peptide and which can induce the production of an antibody against IL-23, wherein the B-cell receptor antigen peptide is represented by formula (I): X1-X2-X3-X4-X5-X6-X7-X8 (in the formula: X1 is S, A, G, T, K or R; X2 is P, A, G, S, T, K or R; X3 is S, A, G, T, K or R; X4 is Q, A, G, T or N; X5 is P, A, G, S, T, Q or N; X6 is W, A, Y or F; X7 is Q, A, G, T or N; and X8 is R, A, G or K).

Description

Vaccine composition for inducing anti-IL-23 antibodies

[0001] The present invention relates to a vaccine composition, and more particularly to a vaccine composition capable of inducing antibodies against IL-23 in vivo.

[0002] Chronic inflammation is known to be involved in the progression of a wide range of chronic diseases, and it has been reported that IL-23 is the factor that initiates chronic inflammation (Non-Patent Documents 1 and 2).

[0003] IL-23 is a heterodimer consisting of two subunit proteins, p19 and p40, and is classified as a pro-inflammatory cytokine. IL-23 is involved in inducing the proliferation of memory T cells and also contributes to promoting and stabilizing the differentiation of naive T cells into Th17 cells. Th17 cells differentiated and stabilized by IL-23 secrete multiple cytokines and inflammatory effectors, enhancing chronic inflammation.

[0004] Deregulation of the Th17 / IL-23 pathway has been reported to be associated with numerous diseases (e.g., psoriasis, cancer, rheumatoid arthritis, systemic lupus erythematosus, diabetes, atherosclerosis, inflammatory bowel disease, multiple sclerosis, Alzheimer's disease, etc.) (Non-Patent Document 3).

[0005] As IL-23 is involved in the onset and progression of various diseases through chronic inflammation, drug discovery research targeting IL-23 as a therapeutic target is being actively pursued, and a relatively large amount of knowledge is being accumulated regarding psoriasis.

[0006] Psoriasis is a chronic inflammatory skin disease caused by abnormalities in Th17 cells. In psoriasis, Th17 cells are stimulated by IL-23, which then releases the inflammatory cytokines IL-17 and IL-22, triggering an inflammatory response. The prevalence of psoriasis is estimated to be 0.9-11.43% (WHO Report, 2016). While most psoriasis patients have mild to moderate disease, severe cases suffer from serious skin symptoms and complications such as psoriatic arthritis, resulting in extremely poor quality of life for these patients.

[0007] In the treatment of mild to moderate psoriasis, topical ointments containing steroids or vitamin D analogs are used. When standard treatments are ineffective, biologics such as anti-TNF-α antibodies, anti-IL-17 antibodies, or anti-IL-23 antibodies are administered. Anti-IL-23 antibody drugs, in particular, have been clinically proven to be highly effective. These include ustekinumab (trade name Stelara®), which targets the p40 subunit, and risankizumab (trade name Skyrizi®), guselkumab (trade name Tremfya®), and tildrakizumab (trade name Ilumya®), which target the p19 subunit. Risankizumab has been reported to achieve a PASI90 (near-complete clearance of psoriatic lesions) rate approximately twice that of ustekinumab, indicating that the molecular target is shifting from p40 to p19.

[0008] However, antibody drugs require large investments in biomanufacturing facilities, resulting in high production costs. Furthermore, because a relatively large amount of antibody needs to be administered per treatment, the drug price is very high, limiting patient access and placing a strain on healthcare finances. To address these issues with antibody drugs, progress is being made in the development of "peptide vaccines" that can induce desired antibodies in the body of the treatment target, and peptide vaccines capable of inducing anti-IL-23 antibodies are also being considered for the treatment of psoriasis.

[0009] For example, it has been reported that a peptide vaccine containing a peptide fragment of the junction region between α-loop C and α-loop D of the mouse IL-23 p19 subunit was effective in mouse models of collagen-induced arthritis (CIA) and TNBS-induced colitis (Non-Patent Documents 4 and 5). Furthermore, Patent Document 1 conducted detailed studies using various peptide fragments contained in the corresponding region of the human IL-23 p19 subunit and identified them as B cell epitopes capable of inducing anti-IL-23 neutralizing antibodies. However, these peptide vaccines use proteins such as keyhole limpet hemocyanin (KLH) or virus-like particles (VLPs) as carriers to enhance immunogenicity, which poses problems such as high production costs and the risk of inducing anti-carrier antibodies, and further improvements are needed. Furthermore, in order to induce sufficient antibody production, activation of helper T cells and natural immunity is necessary. For this reason, conventional peptide vaccines are usually used in combination with adjuvants such as Freund's incomplete adjuvant (IFA) and aluminum salts (Alum), which poses safety risks.

[0010] WO2016 / 193405

[0011] Christina H Liu et al., Nat Immunol. 2017 Oct 18;18(11):1175-1180.Yoichiro Iwakura et al., J Clin Invest. 2006 May;116(5):1218-22.Sarah L Gaffen et al., Nat Rev Immunol. 2014 Sep;14(9):585-600.Rojo A Ratsimandresy et al., Vaccine. 2011; 29:9329-9336.Qingdong Guan et al., Immunotherapy. 2013; 5:1313-1322.

[0012] In light of the above-mentioned background, an objective of the present invention is to provide a novel peptide vaccine that has sufficient antigenicity in vivo and can efficiently induce anti-IL-23 antibodies without conjugation with a carrier protein or the use of an adjuvant.

[0013] As a result of extensive 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 with a B cell receptor antigen peptide having a specific amino acid sequence in the IL-23 p19 protein (corresponding to positions 151-158 of human p19; positions 152-159 of mouse p19) or a partially modified sequence thereof can induce anti-IL-23 neutralizing antibodies in vivo with high efficiency. According to Patent Document 1, a peptide vaccine containing a peptide fragment consisting of the amino acid sequence of positions 152-159 of human p19, which is shifted one amino acid toward the C-terminus (hence, 7 of 8 amino acids are common), did not induce inhibitory activity against anti-IL-23 function (promotion of IL-17 secretion), so the above-mentioned results are quite surprising. Furthermore, the present inventors have confirmed that administration of the peptide vaccine significantly improves symptoms associated with enteritis in a mouse colitis model. Based on these findings, the present inventors conducted further research and completed the present invention.

[0014] That is, the present invention is as follows: Item [1] A vaccine composition capable of inducing the production of antibodies against IL-23, comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, wherein the B cell receptor antigen peptide is represented by the following formula (I): X1-X2-X3-X4-X5-X6-X7-X8(I), wherein X1 is S, A, G, T, K, or R, X2 is P, A, G, S, T, K, or R, X3 is S, A, G, T, K, or R, X4 is Q, A, G, T, or N, X5 is P, A, G, S, T, Q, or N, X6 is W, A, Y, or F, X7 is Q, A, G, T, or N, and X8 is R, A, G, or K. Item [2] The vaccine composition of Item [1], wherein the N-terminal amino acid of the complex is acetylated and / or the C-terminal amino acid of the complex is amidated. Item [3] The vaccine composition of Item [1] or [2], wherein X6 in the B cell receptor antigen peptide is W. Item [4] The vaccine composition of any one of Item [1] to [3], wherein X7 in the B cell receptor antigen peptide is Q. Item [5] The vaccine composition of any one of Item [1] to [4], wherein X8 in the B cell receptor antigen peptide is R. Item [6] The vaccine composition of any one of Item [1] to [5], wherein the B cell receptor antigen peptide comprises the amino acid sequence represented by any one of SEQ ID NOs: 2, 6 to 14, and 17 to 26. Item [7] The vaccine composition of any one of Item [1] to [6], wherein the T cell receptor antigen peptide comprises the amino acid sequence represented by SEQ ID NO: 38. Item [8] The vaccine composition according to any one of items [1] to [7], wherein the complex is linked between the C-terminus of the T cell receptor antigen peptide and the N-terminus of the B cell receptor antigen peptide. Item [9] The vaccine composition according to any one of items [1] to [8], wherein the T cell receptor antigen peptide and the B cell receptor antigen peptide are linked via a linker. Item

[10] The vaccine composition according to item [9], wherein the linker is an amino acid or a peptide. Item

[11] The vaccine composition according to any one of items [1] to

[10] , which does not contain an additive as an adjuvant.Item

[12] The vaccine composition according to any one of items [1] to

[11] , for treating or preventing an IL-23-associated disease. Item

[13] The vaccine composition according to item

[12] , wherein the IL-23-associated disease is selected from the group consisting of psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, diabetes (preferably type 1 diabetes), atherosclerosis, inflammatory bowel disease (IBD) / Crohn's disease, multiple sclerosis, Behçet's disease, ankylosing spondylitis, Vogt-Koyanagi-Harada disease, chronic granulomatous disease, hidradenitis suppurativa, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, neurodegenerative diseases, atopic dermatitis, graft-versus-host disease, and cancer. Item [1A] A method for inducing the production of antibodies against IL-23 in a subject, comprising administering to the subject an effective amount of a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, wherein the B cell receptor antigen peptide is represented by the following formula (I): X1-X2-X3-X4-X5-X6-X7-X8(I), wherein X1 is S, A, G, T, K or R, X2 is P, A, G, S, T, K or R, X3 is S, A, G, T, K or R, X4 is Q, A, G, T or N, X5 is P, A, G, S, T, Q or N, X6 is W, A, Y or F, X7 is Q, A, G, T or N, and X8 is R, A, G or K. Item [1B] A conjugate of a T cell receptor antigen peptide and a B cell receptor antigen peptide for use in inducing the production of an antibody against IL-23, wherein the B cell receptor antigen peptide is represented by the following formula (I): X1-X2-X3-X4-X5-X6-X7-X8(I), wherein X1 is S, A, G, T, K or R, X2 is P, A, G, S, T, K or R, X3 is S, A, G, T, K or R, X4 is Q, A, G, T or N, X5 is P, A, G, S, T, Q or N, X6 is W, A, Y or F, X7 is Q, A, G, T or N, and X8 is R, A, G or K.Item [1C] Use of a conjugate of a T cell receptor antigen peptide and a B cell receptor antigen peptide in the manufacture of a medicament for treating or preventing an IL-23-associated disease, wherein the B cell receptor antigen peptide is represented by the following formula (I): X1-X2-X3-X4-X5-X6-X7-X8(I), wherein X1 is S, A, G, T, K or R, X2 is P, A, G, S, T, K or R, X3 is S, A, G, T, K or R, X4 is Q, A, G, T or N, X5 is P, A, G, S, T, Q or N, X6 is W, A, Y or F, X7 is Q, A, G, T or N, and X8 is R, A, G or K. Item

[14] A B cell receptor antigen peptide represented by the following formula (I): X1-X2-X3-X4-X5-X6-X7-X8(I), wherein X1 is S, A, G, T, K, or R, X2 is P, A, G, S, T, K, or R, X3 is S, A, G, T, K, or R, X4 is Q, A, G, T, or N, X5 is P, A, G, S, T, Q, or N, X6 is W, A, Y, or F, X7 is Q, A, G, T, or N, and X8 is R, A, G, or K. Item

[15] The B cell receptor antigen peptide according to Item

[14] , wherein X6 in formula (I) is W. Item

[16] The B cell receptor antigen peptide according to item

[14] or

[15] , wherein, in formula (I), X7 is Q. Item

[17] The B cell receptor antigen peptide according to any one of items

[14] to

[16] , wherein, in formula (I), X8 is R.

[0015] According to the present invention, the production of anti-IL-23 antibodies can be induced in vivo very efficiently without the use of carrier proteins or adjuvants, thereby enabling inexpensive and safe treatment and / or prevention of IL-23-associated diseases.

[0016] FIG. 1(a) is a schematic diagram of each AJP001 conjugated peptide used in Example 1. FIG. 1(b) is a graph showing the antibody titer of antisera collected from mice administered with each AJP001 conjugated peptide 5 weeks after the initial administration of the peptide in Example 1. FIG. 1(c) is a graph showing the binding ability to rmIL-23 of sera collected from mice administered with each AJP001 conjugated peptide 5 weeks after the initial administration of the peptide in Example 1. FIG. 2 is a graph showing the results of a mouse immunogenicity evaluation test using the AJP001 conjugated peptide (SEQ ID NO: 2). FIG. 2(a) is a graph showing the antibody titer (GMT) of sera collected over time from mice administered with the AJP001 conjugated peptide (SEQ ID NO: 2). Figure 2(b) is a graph showing the binding ability of serum collected from mice administered the AJP001 conjugated peptide (SEQ ID NO: 2) at 6 weeks after the initial administration to rmIL-23. Figures 2(c) and (d) show the results of T cell activation evaluation (number of IFN-γ-positive cells and number of IL-4-positive cells) in mice administered the AJP001 conjugated peptide (SEQ ID NO: 2). Figure 3 shows the results of a drug efficacy evaluation test on a mouse psoriasis model using the AJP001 conjugated peptide (SEQ ID NO: 2) in Example 3. Figure 3(a) is a graph showing the antibody titer of serum collected from hairless rats administered the AJP001 conjugated peptide (SEQ ID NO: 2) at 6 weeks after the initial administration. Figure 3(b) is a graph showing the ear inflammatory response (increase in ear thickness and inflammatory changes in tissue) over time in the group administered the AJP001 conjugated peptide (SEQ ID NO: 2) and the vehicle control group. Figure 3(c) is a photograph of an HE-stained specimen of an ear prepared under each condition. Figure 3(d) is a graph showing the results of microscopic observation of the HE-stained specimen of an ear prepared under each condition, and scoring for inflammatory cell infiltration, edema, and thickening. Figure 4 is a diagram showing the results of a drug efficacy evaluation test on an IL-23-induced mouse ear inflammation model using AJP001 conjugated peptide (SEQ ID NO: 2) in Example 4. Figure 4(a) is a diagram showing the change in ear thickness over time under each condition.Figure 4(b) is a graph showing the mRNA expression of IL-17A, IL-22, IL-1β, LCN-2, and CXCL-2 in the auricle under each condition. Figure 5 shows the results of a mouse immunogenicity evaluation test using the AJP001 conjugated peptide (SEQ ID NO: 6) in Example 6. Figure 5(a) is a graph showing the antibody titer (GMT) of serum collected over time from mice administered the AJP001 conjugated peptide (SEQ ID NO: 6). Figure 5(b) is a graph showing the binding ability to rhIL-23 of serum collected 6 weeks after the initial administration from mice administered the AJP001 conjugated peptide (SEQ ID NO: 6). Figures 5(c) and (d) show the results of T cell activation evaluation (number of IFN-γ-positive cells and number of IL-4-positive cells) in mice administered the AJP001 conjugated peptide (SEQ ID NO: 6). Figure 6 shows the results of a drug efficacy evaluation test in Example 7 using the AJP001 conjugated peptide (SEQ ID NO: 6) on an IL-23-induced mouse ear inflammation model. Figure 6(a) shows the change in ear thickness on days 3 and 4 under each condition. Figure 6(b) is a graph showing the mRNA expression of IL-17A, IL-22, IL-1β, LCN-2, and CXCL-2 in the ear under each condition. Figure 7 shows the results of a monkey immunogenicity evaluation test in Example 8 using the AJP001 conjugated peptide (SEQ ID NO: 6). Figure 7(a) is a graph showing the antibody titer (GMT) of serum collected over time from cynomolgus monkeys administered the AJP001 conjugated peptide (SEQ ID NO: 6). Figure 7(b) is a graph showing the binding ability to rhIL-23 and rmIL-23 of serum collected from cynomolgus monkeys administered the AJP001 conjugate peptide (SEQ ID NO: 6) 6 weeks after the initial administration. Figure 7(c) is a graph evaluating the neutralizing activity of IgG derived from cynomolgus monkeys administered the AJP001 conjugate peptide (SEQ ID NO: 6). Figure 7(d) is a graph showing the results of epitope mapping of anti-IL-23 antibodies induced by the AJP001 conjugate peptide (SEQ ID NO: 6).Figure 8-1 shows a diagram illustrating a test for evaluating the efficacy of the AJP001 conjugated peptide (SEQ ID NO: 2) in a TNBS-induced mouse colitis model in Example 9. Figures 8(a) and 8(b) show the results of ELISA assays of antibody titers (GMT) and binding to mIL-23 in sera obtained from mice administered the AJP001 conjugated peptide (SEQ ID NO: 2) and then inducing colitis with TNBS (at Day 10 relative to TNBS administration). Figure 8-2 shows a diagram illustrating a test for evaluating the efficacy of the AJP001 conjugated peptide (SEQ ID NO: 2) in a TNBS-induced mouse colitis model in Example 9. (d) is a diagram and photograph showing the results of measuring the length (cm) of the large intestine of mice administered with the AJP001 conjugate peptide (SEQ ID NO: 2) and then inducing colitis with TNBS on Day 10. FIG. 8-3 is a diagram showing a drug efficacy evaluation test on a TNBS-induced mouse colitis model using the AJP001 conjugate peptide (SEQ ID NO: 2) in Example 9. (e) is a diagram showing the results of measuring the mRNA expression of various cytokines in colon tissue 2 cm from the anus on Day 10 of mice administered with the AJP001 conjugate peptide (SEQ ID NO: 2) and then inducing colitis with TNBS. FIG. 8-4 is a diagram showing a drug efficacy evaluation test on a TNBS-induced mouse colitis model using the AJP001 conjugate peptide (SEQ ID NO: 2) in Example 9. (f) is a diagram and photographs showing the results of pathological examination of colon tissues 2 to 4 cm from the anus on Day 10 of mice that were administered the AJP001 conjugated peptide (SEQ ID NO: 2) and then had colitis induced with TNBS.

[0017] The present invention will be described in detail below.

[0018] 1. Vaccine Composition The present invention provides a vaccine composition (hereinafter, sometimes referred to as the "vaccine composition of the present invention") that comprises a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide and is capable of inducing the production of antibodies against IL-23 (and is therefore suitable for treating or preventing IL-23-associated diseases).

[0019] 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 (-COO － ), amide (-CONH ２ ) or an ester (—COOR).

[0020] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. １－６ Alkyl group; for example, C cyclopentyl, cyclohexyl, etc. ３－８ Cycloalkyl groups such as phenyl and α-naphthyl ６－１２ Aryl groups; for example, phenyl-C such as benzyl and phenethyl １－２ Alkyl group; α-naphthyl-C such as α-naphthylmethyl １－２ C such as alkyl group ７－１４ Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0021] When the peptide conjugate has a carboxyl group (or carboxylate) other than that at the C-terminus, the carboxyl group may be amidated or esterified, and the ester may be, for example, the C-terminal ester described above.

[0022] Furthermore, the peptide conjugate may contain a protecting group (e.g., a C group such as a formyl group or an acetyl group) in which the amino group of the N-terminal amino acid residue is protected. １－６ C such as alkanoyl １－６those 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 an appropriate protecting group (e.g., C-protecting group such as formyl group, acetyl group, etc.); １－６ C such as alkanoyl group １－６ In one embodiment, the amino group of the N-terminal amino acid residue of the peptide conjugate is acetylated and / or the carboxyl group of the C-terminal amino acid residue is amidated.

[0023] 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.

[0024] The B cell receptor antigen peptide in the vaccine composition of the present invention is a peptide comprising an amino acid sequence represented by formula (I): X1-X2-X3-X4-X5-X6-X7-X8, or a peptide consisting of said 8 amino acids, wherein X1 is S (serine), A (alanine), G (glycine), T (threonine), K (lysine) or R (arginine), X2 is P (proline), A (alanine), G (glycine), S (serine), T (threonine), K (lysine) or R (arginine), X3 is S (serine), A (alanine), G (glycine), T (threonine), K (lysine) or R (arginine), X4 is Q (glutamine), A (alanine), G (glycine), T (threonine), or N (asparagine), X5 can be P (proline), A (alanine), G (glycine), S (serine), T (threonine), Q (glutamine) or N (asparagine); X6 can be W (tryptophan), A (alanine), Y (tyrosine) or F (phenylalanine); X7 can be Q (glutamine), A (alanine), G (glycine), T (threonine) or N (asparagine); and X8 can be R (arginine), A (alanine), G (glycine) or K (lysine).

[0025] In one embodiment, X6 in formula (I) can be W.

[0026] In one embodiment, X7 in formula (I) can be Q.

[0027] In one embodiment, X8 in formula (I) can be R.

[0028] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S or A; X4 is Q or A; X5 is P, A, S, T or Q; X6 is W or A; X7 is Q or A; and X8 is R or A.

[0029] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S or A; X4 is Q or A; X5 is P, A, S, T or Q; X6 is W or A; X7 is Q or A; and X8 is R.

[0030] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S; X4 is Q or A; X5 is P, A, S, T or Q; X6 is W or A; X7 is Q or A; and X8 is R.

[0031] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S or A; X4 is Q; X5 is P, A, S, T or Q; X6 is W or A; X7 is Q or A; and X8 is R.

[0032] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S or A; X4 is Q or A; X5 is P, A, S, T or Q (provided that when X2 is S, X5 is P, A or Q); X6 is W or A; X7 is Q or A; and X8 is R.

[0033] In one embodiment, formula (I) may be such that: X1 is S; X2 is P, S or T; X3 is S; X4 is Q; X5 is P, S, T or Q (provided that when X2 is S, X5 is P or Q); X6 is W or A; X7 is Q or A; and X8 is R.

[0034] In one embodiment, formula (I) may be such that: X1 is S; X2 is P, A, S or T; X3 is S; X4 is Q or A; X5 is P; X6 is W or A; X7 is Q or A; and X8 is R.

[0035] In one embodiment, the 8 amino acids represented by formula (I) can be any of the amino acid sequences represented by SEQ ID NOs: 2, 6-14 and 17-26.

[0036] 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.

[0037] 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 (ELKLIFLHRLKRLRKRLKRK (SEQ ID NO: 38) developed by the present inventors, see WO2016 / 047763 for details), UBITh peptides (UBITh (registered trademark) 1: ISITEIKGVIVHRIETILF (SEQ ID NO: 39), UBITh (registered trademark) 2: KKKIITITRIITIITTID (SEQ ID NO: 40), UBITh (registered trademark) 3: ISISEIKGVIVHKIETILF (SEQ ID NO: 41), ISITEIRTVIVTRIETILF (SEQ ID NO: 42), see US Patent No. 5,649,492 for details) can be used. No. 9,102,752), Tetanous toxisoid peptide (TTaa830-843 peptide: QYIKANSKFIGITE (SEQ ID NO: 43)), etc. can be used. AJP001 is particularly preferred as a T cell receptor antigen peptide in the present invention because it induces the secretion of IL-β1 and IL-18 through activation of the NLRP3 inflammasome and can also activate the innate immune system by inducing the production of TNF-α and IL-6 via the NF-κB pathway.

[0038] In the vaccine composition of the present invention, a T cell receptor antigen peptide and a B cell receptor antigen peptide bind to form a complex. This bond 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. However, this bond 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 is linked to the end of the other peptide chain, the terminal amino acids may be directly linked by a peptide bond or may be linked via a linker (also referred to herein as a "spacer"). 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 antigen-presenting cells to present helper T cell epitopes in the T cell receptor antigen peptide on free MHC class II molecules. 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. Furthermore, L-amino acids present in natural proteins (e.g., glutamic acid, cysteine, and lysine) 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. PEG linkers further containing one or more selected from glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg), and lysine (Lys) can also be used.

[0039] In one embodiment, the complex formed by linking a B cell receptor antigen peptide and a T cell receptor antigen peptide may further contain additional amino acids. The addition of such amino acids is permissible as long as the desired effect of the vaccine composition of the present invention is achieved. The amino acid sequence to be added is not particularly limited, but examples thereof include tags that facilitate 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.), and immunoglobulin Fc tags. 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.

[0040] The conjugates in the vaccine compositions of the present invention can be produced by solid-phase synthesis (Fmoc and Boc methods) or liquid-phase synthesis according to known general peptide synthesis protocols. When the conjugate is one 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.

[0041] In one embodiment, the peptide conjugate in the vaccine composition of the present invention may be conjugated with a carrier protein to enhance its immunogenicity. Carrier proteins are generally substances that confer immunogenicity by binding to molecules (haptens) that are not immunogenic due to their small molecular weight, and 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 some of its amino acids, immunoglobulins, etc. The carrier protein can be conjugated to the N-terminus or C-terminus of the conjugate in the vaccine composition of the present invention. Conjugation can be achieved by introducing a cysteine ​​residue into the antigen peptide of the present invention and then binding it to the amino group of the carrier protein via the SH group in the side chain of the cysteine ​​(MBS method). Conjugation can also be achieved by bonding amino groups, such as the ε-amino group or α-amino group of lysine residues in proteins (glutaraldehyde method).

[0042] In a preferred embodiment, the complex in the vaccine composition of the present invention is not conjugated to a carrier protein. When the complex is taken up by an antigen-presenting cell, the helper T cell epitope in the T cell receptor antigen peptide is released, presented on an MHC class II molecule, and recognized by a T cell receptor on a helper T cell, activating the helper T cell. The activated helper T cell recognizes a B cell that also presents the helper T cell epitope on an MHC class II molecule, secretes cytokines such as IFN-γ, activates the B cell, and potently promotes the production of antibodies against the B cell receptor antigen peptide (IL-23 p19). Therefore, high antibody production can be induced without the use of a carrier protein. When a carrier protein is used in combination with a peptide vaccine, antibodies specific to the carrier protein may be induced, potentially causing undesired side effects. Furthermore, peptide vaccines conjugated to a carrier protein are undesirable due to their increased production costs.

[0043] In one embodiment, the vaccine composition of the present invention may further contain an adjuvant that is pharmaceutically acceptable and compatible with the active ingredient. Adjuvants are generally substances that nonspecifically enhance the host's immune response, 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, montane, squalane, saponin, etc.

[0044] In a preferred embodiment, the vaccine composition of the present invention is not used in combination with an adjuvant. For example, when a peptide capable of activating the innate immune system, such as AJP001, is used as the T cell receptor antigen peptide, the vaccine composition of the present invention alone is sufficient to induce antibody production, and therefore, the use of an adjuvant in combination is not necessary. The use of an adjuvant may induce unexpected side effects, and avoiding its use reduces safety risks.

[0045] The vaccine composition of the present invention can be provided as a pharmaceutical composition comprising a pharmaceutically acceptable carrier in addition to the complex of the T cell receptor antigen peptide and the B cell receptor antigen peptide.

[0046] Pharmaceutically acceptable carriers may be selected appropriately depending on the dosage form, and examples include, but are not limited to, 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, dispersing agents such as surfactants, diluents such as water and physiological saline, and base waxes.

[0047] The vaccine composition of the present invention can be administered to mammals orally or parenterally. Since the complex of T cell receptor antigen peptide and B cell receptor antigen peptide can be degraded in the stomach, parenteral administration is preferred. Formulations suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions, etc. Formulations 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, bacteriostats, isotonicity agents, etc. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. The formulations can be packaged in unit doses or multiple doses in containers such as ampoules or vials. Alternatively, the active ingredient and pharmaceutically acceptable carrier can be lyophilized and stored in a state that requires only dissolving or suspending in an appropriate sterile vehicle immediately before use.

[0048] 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.

[0049] The recipient of the vaccine composition of the present invention is not particularly limited as long as it is a mammal that can be affected by a disease associated with abnormal enhancement of signal transduction involving IL-23 (hereinafter, sometimes referred to as an "IL-23-associated disease"). Examples of such mammals include rodents such as mice, pets such as dogs, livestock such as pigs, horses, and cows, and primates such as humans, monkeys, orangutans, and chimpanzees, with humans being particularly preferred.

[0050] The dose of the vaccine composition of the present invention varies depending on the subject to be administered, the administration method, the administration form, etc., but typically, per adult, the active ingredient, a complex of a T cell receptor antigen peptide and a 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 if the antibody titer decreases, an additional dose can be administered each time.

[0051] Diseases that can be treated or prevented by the vaccine composition of the present invention are the above-mentioned IL-23-associated diseases, including, but not limited to, psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, diabetes (preferably type 1 diabetes), atherosclerosis, inflammatory bowel disease (IBD) / Crohn's disease, multiple sclerosis, Behçet's disease, ankylosing spondylitis, Vogt-Koyanagi-Harada disease, chronic granulomatous disease, hidradenitis suppurativa, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, neurodegenerative diseases (preferably Alzheimer's disease or multiple sclerosis), atopic dermatitis, graft-versus-host disease, and cancer (preferably esophageal carcinoma, colon cancer, lung adenocarcinoma, small cell carcinoma, or oral squamous cell carcinoma).

[0052] In one embodiment, the vaccine composition of the present invention can be used to treat or prevent psoriasis, psoriatic arthritis, neurodegenerative diseases (particularly Alzheimer's disease), diabetes (particularly type 1 diabetes), atherosclerosis, or IBD. Also, in a particularly preferred embodiment, the vaccine composition of the present invention can be used to treat or prevent psoriasis, psoriatic arthritis, or IBD.

[0053] In one embodiment, the vaccine composition of the present invention may be used in combination with an existing therapeutic agent for an IL-23-related disease. For example, in the treatment of psoriasis, treatment with a highly rapid-acting antibody drug is initiated during the active phase of the disease, and administration of the vaccine composition of the present invention is initiated during the remission phase, thereby achieving both a good balance between the patient's QOL and treatment costs.

[0054] In this specification, "treatment" of a disease may include not only curing the disease, but also remission of the disease and improvement in the severity of the disease.

[0055] Furthermore, "prevention" of a disease herein includes not only preventing the onset of the disease but also delaying the onset of the disease. In addition, "prevention" of a disease herein may also include preventing the recurrence of the disease after treatment or delaying the recurrence of the disease after treatment.

[0056] In addition, the term "vaccine composition" as used herein can also be replaced with "pharmaceutical composition" or "drug."

[0057] 2. Method for Treating or Preventing an IL-23-Associated Disease The present invention also provides a method for treating or preventing an IL-23-associated disease (hereinafter, sometimes referred to as the "method of the present invention"), which comprises administering the vaccine composition of the present invention to a subject suffering from an IL-23-associated disease.

[0058] 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, specific examples of IL-23-associated diseases, etc. are the same as those explained in "1. Vaccine composition of the present invention."

[0059] 3. B Cell Receptor Antigen Peptide The present invention also provides the B cell receptor antigen peptide itself (hereinafter, sometimes referred to as the "B cell receptor antigen peptide of the present invention") that constitutes part of the peptide complex contained in the vaccine composition of the present invention.

[0060] The B cell receptor antigen peptide of the present invention is a peptide comprising an amino acid sequence represented by formula (I): X1-X2-X3-X4-X5-X6-X7-X8, or a peptide consisting of said 8 amino acids, wherein X1 is S (serine), A (alanine), G (glycine), T (threonine), K (lysine) or R (arginine), X2 is P (proline), A (alanine), G (glycine), S (serine), T (threonine), K (lysine) or R (arginine), X3 is S (serine), A (alanine), G (glycine), T (threonine), K (lysine) or R (arginine), X4 is Q (glutamine), A (alanine), G (glycine), T (threonine), or N (asparagine), X5 can be P (proline), A (alanine), G (glycine), S (serine), T (threonine), Q (glutamine) or N (asparagine); X6 can be W (tryptophan), A (alanine), Y (tyrosine) or F (phenylalanine); X7 can be Q (glutamine), A (alanine), G (glycine), T (threonine) or N (asparagine); and X8 can be R (arginine), A (alanine), G (glycine) or K (lysine).

[0061] In one embodiment, X6 in formula (I) can be W.

[0062] In one embodiment, X7 in formula (I) can be Q.

[0063] In one embodiment, X8 in formula (I) can be R.

[0064] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S or A; X4 is Q or A; X5 is P, A, S, T or Q; X6 is W or A; X7 is Q or A; and X8 is R or A.

[0065] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S or A; X4 is Q or A; X5 is P, A, S, T or Q; X6 is W or A; X7 is Q or A; and X8 is R.

[0066] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S; X4 is Q or A; X5 is P, A, S, T or Q; X6 is W or A; X7 is Q or A; and X8 is R.

[0067] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S or A; X4 is Q; X5 is P, A, S, T or Q; X6 is W or A; X7 is Q or A; and X8 is R.

[0068] In one embodiment, formula (I) may be such that: X1 is S or A; X2 is P, A, S or T; X3 is S or A; X4 is Q or A; X5 is P, A, S, T or Q (provided that when X2 is S, X5 is P, A or Q); X6 is W or A; X7 is Q or A; and X8 is R.

[0069] In one embodiment, formula (I) may be such that: X1 is S; X2 is P, S or T; X3 is S; X4 is Q; X5 is P, S, T or Q (provided that when X2 is S, X5 is P or Q); X6 is W or A; X7 is Q or A; and X8 is R.

[0070] In one embodiment, formula (I) may be such that: X1 is S; X2 is P, A, S or T; X3 is S; X4 is Q or A; X5 is P; X6 is W or A; X7 is Q or A; and X8 is R.

[0071] In one embodiment, the 8 amino acids represented by formula (I) can be any of the amino acid sequences represented by SEQ ID NOs: 2, 6-14, and 17-26.

[0072] The B cell receptor antigen peptide of the present invention can be used alone as a peptide vaccine that induces anti-IL-23 antibodies, and in a more preferred embodiment, can be used as a peptide vaccine with higher immunogenicity by binding to a T cell antigen receptor peptide. In a particularly preferred embodiment, the B cell antigen receptor peptide of the present invention can be used in a form conjugated with AJP001, thereby being usable as a peptide vaccine that can efficiently induce anti-IL-23 antibodies.

[0073] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0074] Peptide Synthesis (Fmoc Method) Protected peptide resins were synthesized 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 resulting protected peptide resin, which was then cleaved from the resin and deprotected to obtain crude peptides. This crude peptide was then purified using a reverse-phase HPLC column with 0.1% TFA-H. ２ 0 / CH ３ The target peptide was purified by gradient elution using a CN system. Fractions containing the target compound were collected and lyophilized to obtain the target peptide.

[0075] HPLC analysis of peptides The purity of the synthesized peptides 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, 2.1 m x 250 mm, 5 microns Column temperature: Room temperature Mobile phase A: 0.1% aqueous trifluoroacetic acid Mobile phase B: Acetonitrile Gradient conditions: The concentration of mobile phase B was increased linearly from 5% to 80% over 30 minutes (5 → 80% buffer B in 30 minutes).

[0076] Mass analysis of peptides The masses of the synthesized peptides were measured by MALDI-TOF-MS under the following analytical conditions: MALDI-TOF-MS model: Bruker autoflex speed Matrix: 2,5-Dihydroxybenzoic acid Dissolving solution: Mixed solution of 0.1% aqueous trifluoroacetic acid and acetonitrile

[0077] Example 1: Test for evaluating antibody production using AJP001 conjugated peptide (mouse IL-23) Epitope peptides derived from mouse IL-23 shown in Table 1 (SEQ ID NOS: 1 to 5, FIG. 1(a)) were selected as B cell antigens, and conjugated with the T cell antigen AJP001 (Table 2, SEQ ID NO: 38) and ε-aminocaproic acid (sometimes referred to as "Ahx") as a spacer to prepare complexes (AJP001 conjugated peptides) (production was outsourced to Peptide Institute, Inc.). In this specification, the AJP001 conjugated peptide in which the B cell epitope has the amino acid sequence represented by SEQ ID NO: "X" will be referred to as the "AJP001 conjugated peptide (SEQ ID NO: X)" etc.

[0078]

[0079]

[0080] AJP001 conjugated peptides having any of the B cell epitope sequences of SEQ ID NOS: 1 to 5 were dissolved in physiological saline and administered intradermally to BALB / c mice (female, 6 weeks old, N=5-6) at 0.5 mg / body twice at two-week intervals. Blood samples were collected before administration and 5 weeks after the first administration, and the antibody titers against each AJP001 conjugated peptide and their binding ability to recombinant mouse IL-23 protein (rmIL-23) were measured by ELISA. Specifically, epitope peptides dissolved in carbonate buffer at 10 μg / mL were immobilized on a 96-well plate, which was then blocked with 5% skim milk / PBS. Then, serum serially diluted with 5% skim milk / PBS was added and the plate was left to stand overnight at 4°C. After washing the wells with PBS-T, HRP-labeled anti-mouse IgG antibody (GE Healthcare) 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. ２ SO ４ The reaction was stopped by adding 100 ml of 1000 ml of IL-23 (Antibodies-online GmbH) and the absorbance at 450 nm was measured using a plate reader. The serum dilution factor at which the absorbance reached half of the maximum value (OD = 1.75) was defined as the antibody titer, and the geometric mean antibody titer (GMT) was calculated from the individual values. Binding to IL-23 protein was measured by ELISA in the same manner as for antibody titer, using rmIL-23 (Antibodies-online GmbH) as the solid-phase antigen and goat serum for blocking. The antibody titers and binding activity to rmIL-23 of each AJP001 conjugated peptide are shown in Figures 1(b) and (c). As a result, a significant increase in antibody titer and binding to rmIL-23 were confirmed in the antisera from mice administered the AJP001 conjugated peptide having the B-cell epitope sequence of SEQ ID NO: 2 (AJP001 conjugated peptide (SEQ ID NO: 2)).

[0081] Example 2 Mouse Immunogenicity Evaluation Test Using AJP001 Conjugated Peptide (SEQ ID NO: 2) The AJP001 conjugated peptide (SEQ ID NO: 2) was dissolved in physiological saline and administered intradermally to BALB / c mice (female, 6 weeks old, N=6) at 1 mg / body three times at two-week intervals, and blood was collected every two weeks until the eighth week. The antibody titer (GMT) of all collected sera was measured by ELISA, and the binding ability of the sera at the six-week time point to rmIL-23 was measured by ELISA (same method as in Example 1). The antibody titer at each time point and the binding of the induced antibodies to rmIL-23 are shown in Figures 2(a) and (b), respectively.

[0082] 2(a) and (b), the antibody titer in the antisera of mice administered the AJP001 conjugated peptide (SEQ ID NO: 2) increased from week 2 and continued until week 8. Furthermore, it was confirmed that the antisera at week 6 contained antibodies that bound to rmIL-23.

[0083] Additionally, an ELISPOT assay was performed to evaluate T cell activation. BALB / c mice (female, 6 weeks old, N = 3-4) were administered the AJP001 conjugated peptide (SEQ ID NO: 2) or saline three times at two-week intervals, and spleens were collected 7 weeks after the first administration. The collected spleens were ground using a syringe and cell strainer. They were then washed and suspended in HBSS(-), and red blood cells were removed using ACK Buffer (Thermo Fisher Scientific), and splenocytes were isolated. Using the Mouse IFN-γ ELISpot assay kit and Mouse IL-4 ELISpot assay kit (R&D systems), each capture antibody was immobilized on a 96-well plate. Cells were prepared in RPMI 1640 medium (culture medium) containing 10% FBS, 1% penicillin, streptomycin, and 4 ng / mL rmIL-2 to a density of 2.5 × 10^6 cells / mL and seeded at 100 μL / well. To each well, (1) medium alone, (2) 10 μg / mL of T cell antigen AJP001 (SEQ ID NO: 38) alone, (3) 10 μg / mL of AJP001 conjugated peptide (SEQ ID NO: 2), or (4) 100 ng / mL of PMA / ionomycin (PMA / IM, Sigma) was added to stimulate the splenocytes. The stimulation was continued for 2 days under CO ２ The experiment was carried out in an incubator. Then, a detection antibody was added, and after washing, a colorimetric reaction was carried out. The stained cells were counted under a microscope. The number of IFN-γ and IL-4 positive cells was calculated as the number of cells per 10^6 cells (SFC / 10^6 cells). The results are shown in Figure 2(c) and (d). In Figure 2, "*" indicates significance (P<0.05).

[0084] 2(c) and (d), in splenocytes from mice administered with the AJP001-conjugated peptide (SEQ ID NO: 2), an increase in the number of IFN-γ- and IL-4-positive cells was confirmed by stimulation with (2) 10 μg / mL of the T cell antigen AJP001 (SEQ ID NO: 38) alone, and (3) 10 μg / mL of the AJP001-conjugated peptide (SEQ ID NO: 2). This confirmed the activation of AJP001-conjugated peptide-specific T cells.

[0085] Example 3: Efficacy Evaluation Test of AJP001 Conjugated Peptide (SEQ ID NO: 2) in a Psoriasis Model. AJP001 conjugated peptide (SEQ ID NO: 2) was dissolved in saline and administered intradermally to hairless rats (female, 6 weeks old, N = 6) at 1 mg / body, three times at two-week intervals. Starting six weeks after the initial administration, 2% imiquimod (IMQ) was applied intermittently to the ears four times at daily intervals to create a psoriasis model. A control group was also administered 2% IMQ. Efficacy was evaluated by measuring ear thickness using a thickness gauge (Mitutoyo) from the first day of IMQ application until the eighth day. Histopathological evaluation of the ear tissue was also performed on the eighth day. Specifically, the collected ears were fixed in 10% neutral formalin, and specimens were prepared according to standard procedures and stained with HE. The HE-stained specimens were observed under a microscope, and inflammatory cell infiltration, edema, and thickening were scored on a four-point scale (0: normal, 1: mild, 2: moderate, 3: severe), and the sum of the scores was calculated. The antibody titer (GMT) of the serum at 6 weeks was measured by ELISA (using the same method as in Example 1; the secondary antibody was anti-Rat IgG (GE Healthcare)). The results of the antibody titer, ear thickness, and histological examination of the AJP001 conjugated peptide (SEQ ID NO: 2) are shown in Figure 3 . In Figure 3 , "*" indicates significant (P<0.05) compared to "2% IMQ." "#" indicates significant (P<0.05) compared to "Day 0" in (b) and "Normal (normal group)" in (d).

[0086] As shown in Figure 3, an increase in antibody titer was confirmed in hairless rats administered the AJP001 conjugate peptide (SEQ ID NO: 2) (Figure 3(a)). Furthermore, the group administered the AJP001 conjugate peptide (SEQ ID NO: 2) was confirmed to have an inhibitory effect on the ear inflammatory response (increase in ear thickness and inflammatory changes in tissue) observed in the vehicle control group (Figures 3(b), (c), and (d)).

[0087] Example 4: Efficacy evaluation test of AJP001 conjugated peptide (SEQ ID NO: 2) on an IL-23-induced mouse ear inflammation model AJP001 conjugated peptide (SEQ ID NO: 2) was dissolved in physiological saline and administered intradermally to BALB / c mice (female, 6 weeks old, N=8) at 1 mg / body three times at two-week intervals. Six weeks after the initial administration, rmIL-23 was administered intradermally to the ear to induce inflammation. The initial administration of rmIL-23 was designated Day 0, and administration was performed a total of four times on Days 2, 4, and 7. A control group, rmIL-23 group, to which no peptide was administered, was set up. Ear thickness was measured using a thickness gauge on each day of rmIL-23 administration and on Day 9. Additionally, to measure the mRNA expression of IL-17A, IL-22, IL-1β, LCN-2, and CXCL-2 in the ears, ear samples were collected under the same conditions on day 3 of rmIL-23 administration. A control group without rmIL-23 administration was also included for mRNA evaluation. The collected ear samples were frozen and pulverized under liquid nitrogen, and total RNA was extracted using an RNeasy Fibrous Tissue Kit (Qiagen). Subsequently, cDNA was prepared from the extracted RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biomedical Inc.). TaqMan Gene Expression Assays (S18: Mm02601777_g1; CXCL-2: Mm00436450_m1; IL-17A: Mm00439618_m1; IL-22: Mm01226722_m1; IL-1β: Mm00434228_m1; LCN-2: Mm01324470_m1) were used, and Ct values ​​were measured using an Applied Biosystems 7900HT Fast Real-Time PCR System. mRNA expression levels were calculated using the ΔΔCt method and evaluated as relative values ​​to the normal group. The results of evaluating the efficacy of the AJP001 conjugated peptide (SEQ ID NO: 2) on ear thickness and mRNA expression are shown in Figure 4. In Figure 4, "*" indicates significance (P<0.05) compared to "rmIL-23.""#" means that the difference is significant (P<0.05) compared to "Day 0" in (a) and compared to "Normal" in (b).

[0088] As shown in Figures 4(a) and (b), mice administered with the AJP001 conjugated peptide (SEQ ID NO: 2) were confirmed to have an inhibitory effect on the ear inflammatory response observed in the rmIL-23 group (increased ear thickness and mRNA expression of IL-17A, IL-22, IL-1β, LCN-2, and CXCL-2 in the ear).

[0089] Example 5 Antibody Production and Activity Evaluation Using AJP001 Conjugated Peptide (Human IL-23) Candidate epitope peptides derived from human IL-23 shown in Table 1 (SEQ ID NOS: 6 to 37) were selected as B cell antigens, and conjugated with the T cell antigen AJP001 (Table 2, SEQ ID NO: 38) using ε-aminocaproic acid as a spacer to synthesize a complex (AJP001 conjugated peptide) (synthesis was outsourced to Toray Research Center, Inc.).

[0090] AJP001 conjugated peptides (SEQ ID NOS: 6-37) were dissolved in physiological saline and mixed with an equal amount of 2% Alhydrogel (Invivogen). This mixture was intradermally administered at 0.5 mg / body to BALB / c mice (female, 6 weeks old, N=5-6) three times at two-week intervals. Blood samples were collected before administration and 6 weeks after the first administration, and the antibody titers (GMT) against each epitope sequence and the binding ability to recombinant human IL-23 protein (rhIL-23) (Peprotech) were measured by ELISA (same method as in Example 1). Binding to rhIL-23 was evaluated as the binding ratio relative to normal serum by calculating the ratio of absorbance in wells containing antisera derived from mice administered with each AJP001 conjugated peptide (SEQ ID NOS: 6-37) to that in wells containing normal mouse serum. The neutralizing activity of mouse antisera was also evaluated using its effect on IL-17 production in rhIL-23-stimulated mouse splenocytes as an index. Spleens from normal BALB / c mice (female, 5 weeks old, N=3) were homogenized using a syringe and cell strainer, then washed and suspended in HBSS(-). Erythrocytes were then removed using ACK Buffer (Thermo Fisher Scientific) to isolate splenocytes. Splenocytes were cultured in RPMI 1640 medium (culture medium) containing 10% FBS, 1% penicillin / streptomycin, and 4 ng / mL rmIL-2 at a concentration of 2.5 × 10^6 cells / mL and seeded at 100 μL / well into a 96-well plate. Antisera (final concentration 15-fold) from mice administered with each AJP001 conjugated peptide and rhIL-23 (final concentration 10 ng / mL) were added to the culture medium, and the mixture was incubated at 37°C for 2 hours. After that, 100 μL / well of the mixture was added to a 96-well plate seeded with splenocytes, and the mixture was incubated at 37°C / 5% CO ２The cells were cultured in an incubator for 3 days. Control wells were prepared, including untreated wells and wells supplemented with normal mouse serum and rhIL-23. IL-17A / F in the culture supernatant was measured using a Mouse IL-17A / F ELISA kit (R&D systems). The neutralizing activity was evaluated by calculating the percentage of IL-17 relative to normal serum using the following formula based on the IL-17 concentrations in the untreated wells (A), control wells (B), and wells supplemented with antisera from mice administered with each AJP001-conjugated peptide (C).

[0091] Percentage of IL-17 relative to normal serum (%) = 100 × (C-A / B-A)

[0092] The antibody titer, binding to rhIL-23, and neutralizing activity of each AJP001 conjugated peptide are shown in Table 3.

[0093]

[0094] As shown in Table 3, an increase in antibody titer and binding to IL-23 were confirmed in the antisera from mice administered with each AJP001 conjugated peptide. Furthermore, inhibition of IL-17 production (neutralizing activity) was confirmed for the antisera administered with each sequence.

[0095] Example 6: Mouse immunogenicity evaluation test using AJP001 conjugated peptide (SEQ ID NO: 6) The AJP001 conjugated peptide (SEQ ID NO: 6) was dissolved in physiological saline and administered intradermally to BALB / c mice (female, 6 weeks old, N=6) at 1 mg / body three times at two-week intervals, and blood was collected every two weeks until the eighth week. The antibody titer (GMT) of all collected sera was measured by ELISA, and the binding ability of the 6-week serum to rhIL-23 was measured by ELISA. The results of the antibody titer and binding to rhIL-23 of the AJP001 conjugated peptide (SEQ ID NO: 6) are shown in Figures 5(a) and (b). In Figure 5, "*" indicates P<0.05.

[0096] 5(a) and (b), the antibody titer in the antisera from mice administered the AJP001 conjugated peptide (SEQ ID NO: 6) increased from week 2 and continued until week 8. Furthermore, binding to rhIL-23 was confirmed in the antisera 6 weeks later.

[0097] In addition, AJP001-conjugated peptide (SEQ ID NO: 6) or saline was administered three times at two-week intervals to BALB / c mice (female, 6 weeks old, N = 3-4), and spleens were collected 7 weeks after the first administration. ELISPOT assays were performed on the collected spleens using a Mouse IFN-γ ELISpot assay kit and a Mouse IL-4 ELISpot assay kit (R&D systems). The results are shown in Figures 5(c) and (d).

[0098] 5(c) and (d), in splenocytes derived from mice administered with the AJP001-conjugated peptide (SEQ ID NO: 6), stimulation with 10 μg / mL of the T cell antigen AJP001 (SEQ ID NO: 38) and 10 μg / mL of the AJP001-conjugated peptide (SEQ ID NO: 6) confirmed an increase in the number of IFN-γ- and IL-4-positive cells, confirming the activation of AJP001-conjugated peptide-specific T cells.

[0099] Example 7: Efficacy Evaluation Test of AJP001 Conjugated Peptide (SEQ ID NO: 6) in an IL-23-Induced Mouse Ear Inflammation Model. The AJP001 conjugated peptide (SEQ ID NO: 6) was dissolved in physiological saline and administered at 1 mg / body to BALB / c mice (female, 6 weeks old, N=8) three times at two-week intervals. Six weeks after the initial administration, rhIL-23 was administered intradermally to induce inflammation. rhIL-23 was administered daily a total of four times. A control group, the rhIL-23 group, was administered without the AJP001 conjugated peptide. Three and four days after rhIL-23 administration, ear thickness was measured using a thickness gauge. Furthermore, mRNA expression of IL-17A, IL-22, IL-1β, LCN-2, and CXCL-2 in the ear was measured. The results of evaluating the efficacy of the AJP001 conjugated peptide (SEQ ID NO: 6) on ear thickness and mRNA expression are shown in Figure 6. In Figure 6, "*" indicates significant (P<0.05) compared to "rhIL-23." "#" indicates significant (P<0.05) compared to "Normal (normal group)."

[0100] As shown in Figure 6, in mice administered with the AJP001 conjugated peptide (SEQ ID NO: 6), a suppressive effect was confirmed on the ear inflammatory response observed in the rhIL-23 group (increased ear thickness and mRNA expression of IL-17A, IL-22, IL-1β, LCN-2, and CXCL-2 in the ear).

[0101] Example 8: Immunogenicity evaluation test for monkeys using AJP001 conjugated peptide (SEQ ID NO: 6) The AJP001 conjugated peptide (SEQ ID NO: 6) was dissolved in physiological saline and administered subcutaneously to cynomolgus monkeys (female, 4-5 years old, N=4) at 5 mg / body three times at two-week intervals. Blood samples were collected every two weeks from the first day of administration until the 16th week, and the antibody titer (GMT) and binding to IL-23 were measured for the serum at each time point by ELISA (using the same method as in Example 1; anti-monkey IgG (Abcam) was used as the secondary antibody). The results are shown in Figure 7. In Figure 7, "*" indicates P<0.05.

[0102] As shown in Figure 7(a), the antibody titer increased from 2 weeks after the first administration and continued to increase until week 16. Furthermore, the monkey antiserum 6 weeks after the first administration exhibited the ability to bind to rhIL-23, but no binding to rmIL-23 was observed (Figure 7(b)).

[0103] Next, IgG was purified from the monkey antiserum 6 weeks after the initial administration using Protein G HP spin trap (GE Healthcare), and the effect on IL-17 production when mouse splenocytes were stimulated with rhIL-23 was evaluated (using the same method as in Example 5). The results are shown in Figure 7(c).

[0104] As shown in FIG. 7(c), the production of IL-17 was suppressed by adding 300 μg / mL of IgG derived from monkey antiserum.

[0105] Furthermore, epitope analysis was performed using monkey antiserum obtained with the AJP001 conjugated peptide (SEQ ID NO: 6). A 96-well plate onto which the AJP001 conjugated peptide (SEQ ID NO: 6) was immobilized was blocked with goat serum. The AJP001 conjugated peptides (SEQ ID NOs: 6-14) were added at 100 μg / mL to the monkey serum derived from the AJP001 conjugated peptide (SEQ ID NO: 6), and the mixture was incubated overnight at 4°C. The reaction solution was added to the immobilized plate and allowed to stand overnight at 4°C. Control wells were added with only monkey serum derived from the AJP001 conjugated peptide (SEQ ID NO: 6), and untreated wells were also set up. After washing the wells with PBS-T, HRP-labeled anti-monkey IgG antibody (Abcam) diluted with 5% skim milk / PBS was added and the plate was shaken at room temperature for 3 hours. After washing the wells with PBS-T, TMB solution was added and left to stand for 30 minutes in the dark. ２ SO ４ The reaction was stopped by adding 100 ml of 10 ...

[0106] Bonding rate (%) = 100 - [100 × (C-B / A-B)]

[0107] The results are shown in Figure 7(d). As shown in Figure 7(d), the antiserum to the AJP001 conjugate peptide (SEQ ID NO: 6) bound to the AJP001 conjugate peptides (SEQ ID NOs: 6 to 11), but the binding rate to the AJP001 conjugate peptides (SEQ ID NOs: 12 to 14) was reduced. This result suggests that the antibody induced by the AJP001 conjugate peptide (SEQ ID NO: 6) recognizes the C-terminal region of the B cell antigen sequence.

[0108] Example 9: Efficacy evaluation test of AJP001 conjugated peptide (SEQ ID NO: 2) on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced mouse colitis model. The AJP001 conjugated peptide (SEQ ID NO: 2) was dissolved in saline and mixed with an equal amount of 1 mg / mL Type-B / K CpG-ODN K3 (Gene Design, Inc., hereinafter sometimes referred to as "CpG"), and the mixture was subcutaneously administered at 1 mg / body three times at two-week intervals to BALB / c mice (female, 6 weeks old, N=10). Six weeks after the initial administration, TNBS dissolved in 30% ethanol was administered by enema at 2 mg / body. The day of the initial TNBS administration was designated Day 0, and on Day 7, TNBS was administered by enema at 3 mg / body to induce colitis. The mice were divided into three groups: a normal group (no TNBS administration) and a 30% EtOH group (saline / TNBS administration) (TNBS administration but no peptide), and a CpG / TNBS group (adjuvant administration). Body weights were measured on days 1, 2, 3, 4, 7, 8, 9, and 10, including the day of TNBS administration. On day 10, mice were euthanized by cardiac blood collection under deep anesthesia. The serum obtained was analyzed for antibody titer (GMT) and binding to mIL-23 by ELISA (using the same method as in Example 1). Colon tissue was also collected, and colon length (cm) was measured. Cytokine mRNA expression was measured in a 2 cm section from the anus, and histopathological examination was performed in a 2- to 4-cm section. For cytokine mRNA expression, total RNA was extracted using an RNeasy Fibrous Tissue Kit (Qiagen) after bead crushing under ice cooling, and then cDNA was prepared from the extracted RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biomedical Inc.).TaqMan Gene Expression Assays (GAPDH: Mm99999915_g1; IL-17A: Mm00439618_m1; IL-17F: Mm00521423_m1; IL-22: Mm01226722_m1; IL-23: Mm00518984_m1; IL-1β: Mm00434228_m1) were used, and Ct values ​​were measured using an Applied Biosystems 7900HT Fast Real-Time PCR System. mRNA expression levels were calculated using the ΔΔCt method and evaluated relative to the normal group. For histopathological examination, the collected colon was fixed in 10% neutral formalin, and then specimens were prepared according to standard methods and stained with HE. The HE-stained specimens were observed under a microscope and scored for inflammatory cell infiltration (Grade: 0-3), depth of colonic tissue damage (Grade: 0-3), and extent of colonic mucosal damage (Grade: 0-4), and the sum of the scores was calculated.

[0109] The antibody titer of the AJP001 conjugated peptide (SEQ ID NO: 2), binding to rmIL-23, body weight transition (rate of change from Day 0), colon length, mRNA expression in colon tissue, and efficacy evaluation results in histopathological examination are shown in Figures 8-1, 8-2, 8-3, and 8-4. In the figures, "#" indicates significant (P<0.05) compared to "0w." "*" indicates significant (P<0.05) compared to "Saline / TNBS" or "CpG / TNBS."

[0110] As shown in the figure, in mice administered with the AJP001 conjugated peptide (SEQ ID NO: 2), an inhibitory effect on colitis (weight loss, shortening of colon length, inflammatory changes in colon tissue, and mRNA expression of IL-17A, IL-22, and IL-1β in the tissue) observed in the saline / TNBS or CpG / TNBS group was confirmed.

[0111] According to the present invention, peptide vaccines for treating and / or preventing various diseases associated with IL-23 can be produced at low cost, and therefore are extremely useful in the field of pharmaceutical production.

[0112] This application is based on Japanese Patent Application No. 2021-199561 (filing date: December 8, 2021) and Japanese Patent Application No. 2022-162795 (filing date: October 7, 2022), the contents of which are incorporated in their entirety herein.

Claims

1. A vaccine composition capable of inducing the production of antibodies against IL-23, comprising a complex of a T cell receptor antigen peptide and a B cell receptor antigen peptide, wherein the B cell receptor antigen peptide is represented by the following formula (I): X1-X2-X3-X4-X5-X6-X7-X8 (I) wherein: X1 is S, A, G, T, K or R; X2 is P, A, G, S, T, K or R; X3 is S, A, G, T, K or R; X4 is Q, A, G, T or N; X5 is P, A, G, S, T, Q or N; X6 is W, A, Y or F; X7 is Q, A, G, T or N; A vaccine composition wherein X8 is R, A, G or K.

2. The vaccine composition according to claim 1, wherein X6 is W in the B cell receptor antigen peptide.

3. The vaccine composition according to claim 1 or 2, wherein X7 in the B cell receptor antigen peptide is Q.

4. The vaccine composition according to claim 1 or 2, wherein X8 in the B cell receptor antigen peptide is R.

5. The vaccine composition according to claim 1 or 2, wherein the B cell receptor antigen peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 2, 6 to 14, and 17 to 26.

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

38.

7. 3. The vaccine composition according to 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.

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

9. 3. The vaccine composition according to claim 1, which does not contain any additive as an adjuvant.

10. The vaccine composition according to claim 1 or 2, for treating or preventing an IL-23-associated disease.

11. The vaccine composition according to claim 10, wherein the IL-23-associated disease is selected from the group consisting of psoriasis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, diabetes (preferably type I diabetes), atherosclerosis, inflammatory bowel disease (IBD) / Crohn's disease, multiple sclerosis, Behcet's disease, ankylosing spondylitis, Vogt-Koyanagi-Harada disease, chronic granulomatous disease, hidradenitis suppurativa, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, neurodegenerative diseases, atopic dermatitis, graft-versus-host disease, and cancer.