WO2011090065A1 - Peptide vaccine - Google Patents

Abstract

Disclosed is a peptide which has a good immunogenicity and is capable of inducing an enhanced antibody productivity and additional administration of which can enhance the production of the target antibody. Specifically disclosed is a peptide comprising the amino acid sequence of a cell-binding motif of a cell adhesion molecule, an amino acid sequence (T) containing the amino acid sequence of a T cell epitope having an established immunological memory, an amino acid sequence (B) containing the amino acid sequence(s) of one or more specific B cell epitopes of an antigen, and the amino acid sequence of a linker peptide that is positioned between the aforesaid amino acid sequences (T) and (B) in such a manner as to link these amino acid sequences, characterized in that the amino acid sequence of said linker peptide is GKK, VKK or VVKK. Also disclosed are a medicinal composition which comprises the aforesaid peptide as the active ingredient and use of the same.

Description

Peptide vaccine

The present invention relates to a peptide that is excellent in immunogenicity and efficiently induces production of an antibody against an antigen, a pharmaceutical composition containing the peptide as an active ingredient, and use thereof. More specifically, the present invention relates to a peptide that efficiently induces the production of an antibody against a specific B cell epitope of human amyloid β peptide (hereinafter abbreviated as “Aβ peptide”), and Aβ containing the peptide as an active ingredient. The present invention relates to a pharmaceutical composition for the prevention and / or treatment of neurodegenerative diseases such as Alzheimer's disease caused by peptides and its use.

When inducing only the production of a specific antibody against a specific antigen in a living body, the purified antigen alone or an immune adjuvant having an activity to enhance antibody production (non-specific stimulation of the immune mechanism) Thus, a method of administering to a living body together with a substance that enhances a specific immune response to an antigen has been generally used. This method is also widely used as the most effective preventive measure against various infectious diseases.

If there is no other effective preventive or therapeutic method, such as viruses that are ineffective with antibiotics or toxins produced by microorganisms, the body will induce antibody production against these viruses and toxins to protect against them. Often this is the only measure. Currently, this method is in practical use. Inactivated vaccines that inactivate pathogenic microorganisms with organic solvents or ultraviolet irradiation to eliminate infectivity and attenuated pathogens to attenuate pathogenicity to living organisms There is a chemical vaccine. These vaccines are administered parenterally by injection with the exception of some poliovirus vaccines. However, both inactivated vaccines and live vaccines may give anaphylactic shock to living bodies. Furthermore, since virus particles themselves or protein macromolecules are used as immunogens, human serum albumin or gelatin may be used as a preparation stabilizer in order to maintain the stability of vaccine preparations. For this reason, mixing of unknown infectious microorganisms cannot be denied to the substances used for these stabilizers, or anaphylactic shock in the body against the stabilizers may occur. Most vaccines are formulated as injections, but vaccine preparations other than injections are also required.

On the other hand, research and development of new vaccines using polypeptides consisting of partial amino acid sequences of inactivated vaccines and live vaccines, which are safer and more effective than inactivated vaccines and live vaccines, are underway all over the world. . Recombinant vaccines and component vaccines typified by hepatitis B virus vaccines are representative examples of the new vaccines. However, the shorter the chain length of a polypeptide to be used as a vaccine, the better, but the shorter the polypeptide, the major histocompatibility antigen (hereinafter abbreviated as “MHC”) of the class II haplotype of the living body. It is difficult to design to cover a wide range of restraint and have sufficient immunogenicity.

As a method to increase the immunogenicity of the antigen during transmucosal administration, the target antigen, cholera toxin, heat-labile enterotoxin derived from Escherichia coli, and a part of these amino acid sequences are substituted and attenuated Is known as an immune adjuvant (Non-patent Document 1). However, in addition to the target antigen, there are problems such as the production of antibodies against cholera toxin used as an immunoadjuvant and heat-labile enterotoxin derived from E. coli, and it has not been put to practical use.

The present inventors examined the provision of a peptide vaccine that can induce the production of a target antibody in a sufficient amount in vivo without using an immunoadjuvant and can be safely administered, and developed a peptide having a novel structural characteristic ( Patent Document 1). That is, Patent Document 1 has an amino acid sequence containing an amino acid sequence of a T cell epitope on the amino terminus (hereinafter abbreviated as “N-terminus”) side, and a carboxy terminus (hereinafter referred to as “linkage peptide”). , Abbreviated as “C-terminal”.) A peptide having an amino acid sequence of a cell binding motif of a cell adhesion molecule is disclosed in a peptide having an amino acid sequence containing an amino acid sequence of a B cell epitope on the side. Peptides having such a structure efficiently enhanced sufficient production of specific antibodies against the target antigen in multiple MHC class II haplotypes when administered transmucosally such as nasally in the absence of immune adjuvant. . In addition, it has been clarified that the peptide is a highly safe polypeptide that has low ability to induce production of antibodies other than the target specific antibody and does not induce side effects such as anaphylaxis. Furthermore, it was clarified that the polypeptide has an immune adjuvant effect.

The present inventors further disclosed that a peptide that efficiently induces the production of antibodies against human Aβ was prepared based on the structural characteristics disclosed in Patent Document 1, and that the present peptide can be used for the treatment of Alzheimer's disease. (Patent Document 2). That is, Patent Document 2 has an amino acid sequence (hereinafter sometimes abbreviated as “T”) containing an amino acid sequence of a T cell epitope for which immunological memory is established on the N-terminal side, and a linker peptide ( Hereinafter, an amino acid sequence (hereinafter abbreviated as “B”) that includes one or more B cell epitope amino acid sequences of the Aβ peptide on the C-terminal side, in some cases, abbreviated as “L”. And the amino acid sequence of the cell binding motif of the cell adhesion molecule is selected from the N-terminal side of T, between T and L, between L and B, and at least one selected from the C-terminal side of B. Peptides having one position are disclosed.

Alzheimer's disease (hereinafter abbreviated as “AD”) is said to occupy many causes of the onset of senile dementia, causing a memory impairment and movement disorder in patients and a marked decrease in quality of life (QOL). It is. In addition to the treatment of illnesses, a large amount of nursing care is required, such as time loss, loss of work opportunities, mental burden, etc. of the patient and their family members, as well as medical costs as well as social burdens. is there. In advanced countries with an aging population, dementia is a serious problem as the number of patients continues to increase.

AD is a type of serious neurodegenerative disease that does not have a fundamental cure, and is pathologically composed of 42 amino acids (SEQ ID NO: 1 in the sequence listing) or 40 amino acids (SEQ ID NO: 2 in the sequence listing). This is a disease characterized by deposition of so-called “senile plaques” in which Aβ accumulates in the central nerve, neurofibrillary tangles, and neuronal degeneration and loss. For the treatment of AD, clinical trials with cholinesterase inhibitors and anti-hyperlipidemic agents and cranial nerve protective agents such as ethyl eicosapentaenoic acid (EPA) are being conducted.

In the development of treatment methods for AD, vaccine development using Aβ, which plays a central role in AD, as a target molecule is also in progress (Non-patent Document 2). Development of new adjuvants such as human anti-Aβ monoclonal antibodies, DNA vaccines, CpGDNA, and peptide vaccines for oral administration is also underway. However, since DNA vaccines are MHC class I-restricted, it is easy to induce the activation of cytotoxic T cells, and it was therefore considered not suitable for the treatment of AD. Since peptide vaccines are weakly immunogenic and have a low ability to induce antibody production against Aβ, an adjuvant is often required to induce antibody production, and an excessive immune reaction may be caused by the use of an adjuvant. In addition, if the antigen is relatively high in molecular weight, it contains a large number of T cell epitopes and B cell epitopes, so that an excessive immune reaction against these epitopes may be induced. Such an excessive immune reaction may cause an unexpected inflammatory reaction, and it is considered that there is a risk of side effects such as encephalitis. Therefore, the fundamental treatment method of AD has not been established yet. Moreover, although Aβ is known to be involved in the onset and exacerbation of various diseases other than AD, methods for preventing and treating these diseases have not been established.

International Publication No. WO 2004/087767 Pamphlet. International Publication No. WO2007 / 097251 pamphlet.

Previously, the present inventors have developed a peptide having a novel structural characteristic that is excellent in immunogenicity, can induce a sufficient amount of a target antibody in vivo without using an immunoadjuvant, and can be safely administered. That is, cell adhesion within a peptide having an amino acid sequence containing the amino acid sequence of the T cell epitope on the N-terminal side and having an amino acid sequence containing the amino acid sequence of the B cell epitope on the C-terminal side across the linker peptide A peptide having an amino acid sequence of a cell-binding motif of a molecule is disclosed (Patent Document 1). In addition, based on such structural characteristics, a peptide that efficiently induces the production of antibodies against Aβ, that is, an amino acid sequence containing an amino acid sequence of a T cell epitope that has established immune memory in the living body on the N-terminal side (T ), An amino acid sequence (B) containing an amino acid sequence of one or more B cell epitopes of Aβ peptide across the linker peptide (L), and a cell adhesion molecule A peptide having an amino acid sequence of a cell binding motif at least one position selected from the N-terminal side of T, between T and L, between L and B, and the C-terminal side of B has been disclosed (Patent Literature) 2). With such a peptide, high anti-Aβ antibody production was observed in normal mice.

An object of the present invention is to provide a peptide having excellent immunogenicity and higher ability to induce antibody production.

Another object of the present invention is to provide a peptide which is excellent in immunogenicity and has a higher ability to induce antibody production, and which can enhance the production of the target antibody by additional administration.

Another object of the present invention is a peptide that is excellent in immunogenicity and has a higher ability to induce antibody production, and the production of the target antibody can be enhanced by additional administration. It is another object of the present invention to provide a peptide that decreases rapidly after the administration of the peptide.

Another object of the present invention is to provide a pharmaceutical composition containing the peptide as an active ingredient.

The present inventors have intensively studied to solve the above problems, and each structural element contained in the Aβ peptide vaccine disclosed in Patent Document 2, that is, an antibody against each of the T cell epitope, linker peptide, and B cell epitope We focused on the amount of production. Accordingly, a linker peptide having low antigenicity was searched, and as a result, the amino acid sequence of the cell binding motif of the cell adhesion molecule, the amino acid sequence of the T cell epitope, the amino acid sequence of the B cell epitope, and GKK, VKK or VVKK as the linker peptide. It has been found that the peptide comprising the amino acid sequence represented by (SEQ ID NO: 3 in the sequence listing) efficiently induces the production of the target antibody. The inventors have further found that the peptide enhances the production of the antibody of interest more than the previous peptide upon additional administration, completing the present invention.

That is, the present invention relates to:
1. A peptide having the amino acid sequence represented by the general formula: R1-T-R2-L-R3-B-R4 or a salt thereof, wherein T is an amino acid sequence including the amino acid sequence of a T cell epitope for which immunological memory is established L represents the amino acid sequence of the linker peptide, B represents the amino acid sequence comprising the amino acid sequence of one or more specific B cell epitopes of the antigen, and at least one of R1, R2, R3 and R4 is cell adhesion A peptide showing the amino acid sequence of a cell-binding motif of a molecule or a salt thereof, wherein the amino acid sequence of the linker peptide is an amino acid sequence represented by GKK, VKK or VVKK, or a salt thereof,
2. The T cell epitope has an amino acid sequence derived from any one or two or more peptides selected from peptides used as antigens for vaccination of diphtheria, tuberculosis, tetanus, and pertussis. Peptide or salt thereof,
3. 1. The B cell epitope has an amino acid sequence including the amino acid sequence of the B cell epitope of Aβ peptide. Or 2. Peptide or salt thereof,
4). The amino acid sequence including the amino acid sequence of the B cell epitope of the Aβ peptide is the first amino acid sequence from the first amino acid to the 15th amino acid sequence (SEQ ID NO: 4 in the sequence listing) from the N terminus of Aβ, or the first amino acid sequence from the N terminus of Aβ. 2. The 13th amino acid sequence from the amino acid (SEQ ID NO: 5 in the sequence listing). Peptide or salt thereof,
5. 4. The amino acid sequence according to any one of 4 to 11, selected from SEQ ID NOs: 6, 11, 13, 43 and 44 in the sequence listing. Peptide or salt thereof,
6). A peptide comprising the amino acid sequence according to any one selected from SEQ ID NOs: 6 to 11, 13, 43 and 44 in the sequence listing, or a salt thereof,
7). A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing or a salt thereof;
8). A peptide comprising the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing or a salt thereof;
9. A peptide comprising the amino acid sequence set forth in SEQ ID NO: 8 in the sequence listing or a salt thereof;
10. A peptide comprising the amino acid sequence set forth in SEQ ID NO: 9 in the sequence listing or a salt thereof;
11. A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing or a salt thereof;
12 A peptide comprising the amino acid sequence set forth in SEQ ID NO: 11 in the sequence listing or a salt thereof,
13. A peptide comprising the amino acid sequence set forth in SEQ ID NO: 13 in the sequence listing or a salt thereof;
14 A peptide comprising the amino acid sequence set forth in SEQ ID NO: 43 in the sequence listing or a salt thereof,
15. A peptide consisting of the amino acid sequence set forth in SEQ ID NO: 44 in the sequence listing or a salt thereof,
16. 1 above. To 15. A pharmaceutical composition comprising as an active ingredient at least one peptide selected from the above or a pharmacologically acceptable salt thereof,
17. 16. The above-mentioned 16. containing one or more pharmaceutically acceptable additives for formulation. A pharmaceutical composition of
18. 3 above. To 15. A pharmaceutical composition capable of inducing production of an antibody against Aβ, comprising one or more pharmaceutically acceptable pharmaceutical additives together with any of the peptides or salts thereof,
19. 16. For the prevention and / or treatment of a disease caused by Aβ. To 18. Any pharmaceutical composition of
20. 16. For the prevention and / or treatment of at least one disease selected from Alzheimer's disease, Alzheimer-type senile dementia, mild cognitive impairment, senile dementia, Down's syndrome and amyloidosis. To 18. Any pharmaceutical composition of
21. 16. For the prevention and / or treatment of Alzheimer's disease To 18. Any pharmaceutical composition of
22. 1 above. To 15. DNA encoding any of the peptides.

According to the present invention, it is possible to provide a peptide that is excellent in immunogenicity, has a high ability to induce antibody production, and can enhance the production of the target antibody by additional administration.

According to the present invention, it is possible to further provide a pharmaceutical composition having an active ingredient a peptide that is excellent in immunogenicity, has a high ability to induce antibody production, and can enhance the production of the target antibody by additional administration. .

Furthermore, according to the present invention, it has excellent immunogenicity, has a high antibody production-inducing ability, can enhance the production of the target antibody by additional administration, and the antibody induced to produce the peptide ends the continuous administration of the peptide. It is also possible to provide a peptide that rapidly decreases later. An antibody whose antibody rapidly decreases after administration is less likely to cause side effects due to an immune reaction such as an excessive inflammatory reaction due to an antigen. In particular, a vaccine against an internal antigen such as Aβ can be a great advantage because it has few such side effects.

It is a figure which shows the result of having evaluated the anti-A (beta) antibody production induction ability after the additional administration of various A (beta) peptide vaccines using the Balb / c mouse. Evaluation was performed using 4 mice per group, and the amount of anti-Aβ antibody was the mean ± S.D. E. It showed in. All of the peptides tested had an amino acid sequence represented by RGD, which is a cell-binding motif of a cell adhesion molecule, at the N-terminus, followed by a T cell derived from diphtheria toxoid (hereinafter abbreviated as “DiTox”). It has a structure in which an amino acid sequence including the amino acid sequence of the epitope, an amino acid sequence of the linker peptide, and an amino acid sequence including the amino acid sequence of the B cell epitope derived from the Aβ peptide are sequentially linked. Among the eight types of peptides evaluated, RGD-DiTox (19) -GKK-Aβ (which has a linker peptide consisting of an amino acid sequence represented by GKK (hereinafter sometimes abbreviated as “GKK linker peptide”) ( 1-15) showed the highest ability to induce anti-Aβ antibody production, and increased production of anti-Aβ antibody was observed even after 6 additional administrations. Other peptides having the GKK linker peptide also showed high anti-Aβ antibody production inducing ability. RGD-DiTox (19) -VVKK-Aβ (1-15) having a linker peptide consisting of an amino acid sequence represented by VVKK (hereinafter sometimes abbreviated as “VVKK linker peptide”) is also highly anti-Aβ It showed the ability to induce antibody production. In the figure, “Veh” indicates the result of administration of phosphate buffered saline (PBS) instead of the Aβ peptide vaccine. Example 1 It is a figure which shows the result of having evaluated the anti-A (beta) antibody production induction ability after the additional administration of various A (beta) peptide vaccines using the Balb / c mouse. In the figure, “Vehicle” indicates the result of administration of phosphate buffered saline (PBS) instead of the Aβ peptide vaccine. “KK1-13” represents RGD-DiTox (20) -KK-Aβ (1-13). “VKK1-13” indicates RGD-DiTox (19) -VKK-Aβ (1-13). “VVKK1-13” indicates RGD-DiTox (19) -VVKK-Aβ (1-13). (Example 3) It is a figure which shows the result of having evaluated the anti-Aβ antibody production induction ability after the additional administration of various Aβ peptide vaccines using JU-Tg2576 mice. In the figure, “Vehicle” indicates the result of administration of phosphate buffered saline (PBS) instead of the Aβ peptide vaccine. “Tcell rev” indicates RGD-DiTox (Rev) -KK-Aβ (1-13). “KK1-13” represents RGD-DiTox (20) -KK-Aβ (1-13). “VKK1-15” represents RGD-DiTox (19) -VKK-Aβ (1-15). “VVKK1-13” indicates RGD-DiTox (19) -VVKK-Aβ (1-13). “VVKK1-15” indicates RGD-DiTox (19) -VVKK-Aβ (1-15). Example 4 It is a figure which shows the amino acid sequence and sequence number of various peptides.

1. Peptide vaccine The present invention relates to a peptide having an amino acid sequence represented by the following general formula or a salt thereof, wherein T represents an amino acid sequence including the amino acid sequence of a T cell epitope for which immunological memory is established, and L represents a linker peptide. A represents an amino acid sequence, B represents an amino acid sequence including the amino acid sequence of one or more specific B cell epitopes of an antigen, and at least one of R1, R2, R3 and R4 is an amino acid of a cell binding motif of a cell adhesion molecule The present invention relates to a peptide having a sequence or a salt thereof, wherein the amino acid sequence of the linker peptide is an amino acid sequence represented by GKK, VKK or VVKK.
(Chemical formula 1)
R1-T-R2-L-R3-B-R4

In this specification, the amino acid sequence is expressed in three letters in the sequence listing, and in one letter in the other descriptions.

The peptide according to the present invention has an ability to induce the production of an antibody against a B cell epitope contained in the peptide or an amino acid sequence containing the peptide, and enhances the production of the target antibody by additional administration of the peptide. be able to. The antibodies referred to in the present invention are mainly immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) antibodies, which are secreted into blood and body fluids, as well as nasal cavity, oral cavity, This naturally includes antibodies secreted into the eye, digestive tract and the like.

In the peptide according to the present invention, the amino acid sequence of the linker peptide arranged so as to link between the amino acid sequence containing the amino acid sequence of the T cell epitope and the amino acid sequence containing the amino acid sequence of the B cell epitope is GKK, VKK Alternatively, the amino acid sequence is represented by VVKK. In the previous disclosure (Patent Documents 1 and 2), examples of the linker peptide include a recognition sequence of a protease involved in antigen processing, particularly a dipeptide, and a dipeptide composed of lysine-lysine (KK), which is a recognition sequence for cathepsin B, is recommended. It had been. However, an Aβ peptide vaccine prepared using a dipeptide composed of KK as a linker peptide sometimes did not increase the amount of anti-Aβ antibody produced when repeated boosting was performed, and the degree of increase was low. On the other hand, an Aβ peptide vaccine prepared using a peptide consisting of an amino acid sequence represented by GKK, VKK or VVKK as a linker peptide is an Aβ peptide vaccine prepared using a dipeptide consisting of KK as a linker peptide, as shown in the Examples described later. In comparison, the anti-Aβ antibody production inducing ability was higher, and the increase in the amount of antibody produced by additional administration was also large. Since the linker site is not a site directly involved in inducing the production of the target antibody, it can be considered that the same result can be obtained by using the B cell epitope of another antigen instead of the B cell epitope of the Aβ peptide. it can. Furthermore, the Aβ peptide vaccine prepared by using a peptide consisting of an amino acid sequence represented by GKK, VKK or VVKK as a linker peptide is compared with an Aβ peptide vaccine prepared using a dipeptide consisting of KK as a linker peptide. The medium anti-Aβ antibody concentration decreases rapidly. As a weakness of a vaccine, once an immune reaction induced by the vaccine is induced, it is not easy to control it or restore it to its original state, which may cause, for example, an excessive inflammatory reaction and side effects. It is generally known that there is. However, the peptide vaccine according to the present invention has a low possibility of inducing side effects because the blood concentration of the target antibody is rapidly reduced after the administration. In particular, a vaccine against an internal antigen such as Aβ has a great advantage that it has few side effects. In addition, the rapid reduction of the blood anti-Aβ antibody concentration after the end of administration of the Aβ peptide vaccine prepared using a peptide consisting of the amino acid sequence represented by GKK, VKK or VVKK as a linker peptide includes an Aβ-derived B cell epitope Even if the length of the amino acid sequence was changed, it was recognized to the same extent. Therefore, it is considered that it does not depend on the difference in the B cell epitope but depends on the difference in the sequence of the linker peptide. Therefore, in the peptide vaccine according to the present invention, even if a B cell epitope of another antigen is used in place of the B cell epitope of Aβ peptide, the blood concentration of the target antibody can be quickly reduced after the administration is completed. it can. Thus, the Aβ peptide vaccine prepared by using a peptide consisting of an amino acid sequence represented by GKK, VKK or VVKK as a linker peptide has a great advantage of having a very high antibody production inducing ability and high safety. An Aβ peptide vaccine prepared by using a peptide consisting of an amino acid sequence represented by GKK as a linker peptide is more preferable because of its higher ability to induce antibody production.

The “epitope” as used in the present invention is also referred to as an antigenic determinant, and means a specific structural unit of an antigen molecule recognized by T cells, B cells and antibodies involved in an immune response, and has a 6 to 10 amino acid sequence. Or a sequence of 5 to 8 monosaccharides. “Antigen” is a general term for substances that stimulate a living body to induce an immune response such as antibody production or cellular immunity. Generally, it refers to a protein that elicits an immune response. Antibodies are produced against various organic substances such as sugars, lipids, and nucleic acids, but substances other than proteins generally exhibit activity as antigens when they are combined with proteins to form complexes. The specific structural units of antigen molecules recognized by T cells and B cells are called “T cell epitopes” and “B cell epitopes”, respectively.

In the peptide according to the present invention, the B cell epitope is used as a site for inducing antibody production. Amino acid sequences of B cell epitopes are antigens such as toxins, allergens, enzymes, cell membrane antigens, tumor-specific antigens derived from microorganisms, cells, or tumor cells, each of which has already been clarified in the literature. The amino acid sequence of a known antigen epitope itself may be used, or various antigens that are actually immunogenic to a living body, or a partial peptide of the antigen is immunized by a conventional method to identify a B cell epitope sequence, A peptide having this identified amino acid sequence may be used as a B cell epitope. In addition, the B cell epitope may be on the same antigen as the T cell epitope described later, and may share a part or all of the amino acid sequence with the B cell epitope. Therefore, the B cell epitope portion is used for infection prevention, prevention and treatment of inflammatory diseases such as cancer, tumor, ulcer, hepatitis, immune diseases such as allergies and atopic dermatitis, neutralization of enzymes, clinical tests, etc. A preferred antigen peptide can be selected depending on the purpose of use of the antibody, such as detection of various antigens used. Peptides containing these B cell epitopes may be used as they are, or may be used in the form of dimers, trimers or multimers linked to tandem. In addition, two or more B cell epitopes existing on the same antigen can be linked in tandem, or the whole antigen can be used as a B cell epitope. In the case of treatment of diseases involving multiple types of antigenic proteins, it is also possible to design peptides that can produce antibodies against multiple types of antigenic proteins with one peptide by linking those B cell epitopes in tandem. it can. In this case, by processing the recognition sequence of the protease involved in the antigen processing described above as a linker peptide between each B cell epitope, the processing of each epitope can be performed more reliably.

In the peptide according to the present invention, a peptide that induces antibody production against Aβ can be produced by using the B cell epitope of Aβ peptide as a site that induces antibody production. The “B cell epitope of Aβ peptide” refers to a peptide comprising the amino acid sequence of the B cell epitope of Aβ that causes Alzheimer's disease. Among them, a peptide having an amino acid sequence that can dissolve Aβ aggregation fibers, inhibit the deposition of Aβ into nerve fibers and other organs, and inhibit the binding to acetylcholine receptor is preferable. From the point of view, a peptide including an amino acid sequence near the N-terminus of Aβ is particularly preferable, and a peptide composed of about 5 to 20 amino acids is preferable. If the number of constituent amino acids is too small, the ability to induce antibody production is lost. If the number of amino acids is too large, antigenicity is increased, but on the other hand, it may contain an epitope that induces the production of antibodies that induce side effects. The peptide containing the B cell epitope may be used as it is, or may be used in the form of a dimer, trimer or multimer linked to tandem. Further, if necessary, two or more B cell epitopes present on Aβ may be linked in tandem. For example, the entire Aβ, for example, 42 amino acids (SEQ ID NO: 1 in the sequence listing, hereinafter “Aβ ( 1-42) ”or 40 (SEQ ID NO: 2 in the sequence listing, hereinafter abbreviated as“ Aβ (1-40) ”) may be used as the B cell epitope. It is also possible to design a peptide that links multiple epitopes and induces the production of multiple types of antibodies simultaneously. In these cases, by processing the recognition sequence of the protease involved in the antigen processing described above as a linker peptide between each B cell epitope, the processing of each epitope can be performed more reliably.

In the peptide according to the present invention, when the amino acid sequence near the N-terminus of Aβ is used as the B cell epitope, it preferably has at least the 4-10th amino acid sequence from the N-terminus of Aβ. A peptide having the amino acid sequence of 1-15th DAEFRHDSGYEVHHQ in the amino acid sequence shown in No. 1 or 2 (SEQ ID No. 4 in the sequence listing, hereinafter abbreviated as “Aβ (1-15)”), 1-13th A peptide having the amino acid sequence of DAEFRHDSGYEVH (SEQ ID NO: 5 in the Sequence Listing, hereinafter abbreviated as “Aβ (1-13)”), a peptide having the amino acid sequence of the 1st to 11th DAEFRHDSGYE (SEQ ID NO: 14 in the Sequence Listing) Hereinafter, abbreviated as “Aβ (1-11)”.) 3-1. Peptide having the first amino acid sequence of EFRHDSGYE (SEQ ID NO: 15 in the Sequence Listing, hereinafter abbreviated as “Aβ (3-11)”), Peptide having the amino acid sequence of 3-15th EFRHDSGYEVHHQ (in the Sequence Listing) SEQ ID NO: 16, hereinafter abbreviated as “Aβ (3-15)”). In addition, as a B cell epitope of the Aβ peptide, a peptide having the amino acid sequence of 1-10th DAEFRHDSGY in the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing (SEQ ID NO: 17 in the sequence listing, hereinafter “Aβ (1- 10) ”)), a peptide having the amino acid sequence of the 1-8th DAEFHRHDS (SEQ ID NO: 18, in the sequence listing, hereinafter abbreviated as“ Aβ (1-8) ”) 1-6th DAEFFRH (SEQ ID NO: 19 in the sequence listing, hereinafter abbreviated as “Aβ (1-6)”). In view of the ability to induce antibody production against Aβ peptide and side effects, Aβ (1-15) or Aβ (1-13) is preferably used, and Aβ (1-15) is particularly preferably used.

The “T cell epitope with which immunological memory is established” used for the peptide according to the present invention means that when the peptide vaccine according to the present invention is administered, the living body to which the peptide is administered has already established immune memory. Say. For example, a peptide containing the same T cell epitope as that of the peptide of the present invention may be administered in advance to a living body to which the peptide of the present invention is administered to establish immune memory. The T cell epitope for which immune memory is established in a living body preferably refers to a T cell epitope for which immune memory is established in a human. In particular, a T cell epitope possessed by an antigenic peptide that has acquired immunity since early childhood by vaccination or the like can be used without performing the above-described operation for establishing immune memory. Further, if the above operation is performed again on a patient who has already established immune memory, further improvement of the effect can be expected.

The T cell epitope binds with an HLA-DR molecule, which is an MHC class II molecule, and its aggregope, and is presented as an antigen for helper T cells. “Agretope” refers to the amino acid residue at the site of binding to the HLA-DR molecule possessed by a T cell epitope. T cell epitopes are restricted to HLA-DR haplotypes, so use duplicate / shifted multi-agretope peptides that are restricted by as many HLA-DR haplotypes (alleles) as possible and amino acid sequences containing them It is preferable to do this.

Examples of T cell epitopes used in the present invention include peptides used as antigens for vaccination of diphtheria, tetanus, pertussis, sublingual adenitis, rubella, measles, tuberculosis, etc. It is preferable because immune memory is established. Inoculation of three mixed vaccines including diphtheria toxoid, tetanus toxoid, pertussis vaccine and BCG (tuberculosis) is mandatory in Japan, and since it is widely practiced worldwide, from the viewpoint of versatility Is particularly preferred.

Since any amino acid sequence of a peptide used as an antigen for vaccination is already known, the amino acid sequence of a known T cell epitope of the peptide may be used. In addition, an epitope sequence of a partial peptide of an antigen may be identified by actually using a human peripheral blood rejuvenation reaction or the like, and a peptide having this identified amino acid sequence may be used as a T cell epitope. In addition, using the available HLA-DR T cell epitope prediction program, it is possible to optionally select a multiaggregate region that binds to many HLA-DRs and determine the amino acid sequence of the peptide. it can.

In addition, it is preferable that the T cell epitope itself has a lower ability to induce antibody production against the epitope from the viewpoint of side effects and the like. On the other hand, if an amino sequence peptide derived from a sensitizing antigen used for vaccination is used as an epitope, the epitope is originally used in humans to induce the production of antibodies against this epitope. Even if an antibody against this epitope is produced, its safety has been established and there is an advantage that side effects are unlikely to occur. These T cell epitope peptides may be used as they are, or the same or multiple types of T cell epitopes may be linked in tandem. T cell epitopes can also function even when amino acid residues other than the amino acid residues that become an agretope are replaced with other amino acid residues.

Specifically, as this T cell epitope, for example, a peptide having the amino acid sequence of AYNFVESIINLFQVVHNSYN, which is an epitope derived from diphtheria toxoid (SEQ ID NO: 20 in the sequence listing) (hereinafter abbreviated as “DiTox (20)”). , A peptide having the amino acid sequence of AYNFVESIINLFQVVHNSY (SEQ ID NO: 21 in the Sequence Listing) (hereinafter abbreviated as “DiTox (19)”), and a peptide having the amino acid sequence of YNFVESIINLFQVVHNSYN (SEQ ID NO: 22 in the Sequence Listing), and A peptide having the amino acid sequence of LQTMVKLFNRINKNNVA (SEQ ID NO: 23 in the sequence listing), an epitope derived from tetanus toxoid, FLQTMVKLFNRIKNNVAG A peptide having a amino acid sequence (SEQ ID NO: 24 in the sequence listing) (hereinafter abbreviated as “TetT1L”), a peptide having an amino acid sequence of IHVLHGLYGMQVSSHE (SEQ ID NO: 25 in the sequence listing), and a peptide having an amino acid sequence of LIHVVLHGLYGMQVSSHEI (SEQ ID NO: 26 in the Sequence Listing), peptide having the amino acid sequence of YIVNEDKFQILYNSIMYG (SEQ ID NO: 27 in the Sequence Listing), peptide having the amino acid sequence of QYIVNEDKFQILYNSIMYGF (SEQ ID NO: 28 in the Sequence Listing) (hereinafter abbreviated as “TetT3L)” . ), A peptide having the amino acid sequence of SYQMYRSLEYQVDA (SEQ ID NO: 29 in the sequence listing), a peptide having the amino acid sequence of RSYQMYRSLEYQVDAI (SEQ ID NO: 30 in the sequence listing), a peptide having the amino acid sequence of NINIFMESSRSFLV (SEQ ID NO: 31 in the sequence listing), Peptide having the amino acid sequence of ININFMRESSRSFLVN (SEQ ID NO: 32 in the Sequence Listing), peptide having the amino acid sequence of IQMSDPAYNINISLPSYYPD, a peptide derived from MPT64 known as a secretory protein common to Mycobacterium tuberculosis / bovis (Mycobacterium tuberculosis / bovis) SEQ ID NO: 33 in the sequence listing) (hereinafter, “MptL (43-62)” Abbreviation), a peptide having the amino acid sequence of IQMSDPAYNINISLPS (SEQ ID NO: 34 in the sequence listing), a peptide having the amino acid sequence of DPAYNINISLPSYPD (SEQ ID NO: 35 in the sequence listing) and a peptide having the amino acid sequence of YNINISLPSYPDQKS (SEQ ID NO: in the sequence listing) And an epitope having an amino acid sequence such as 36). Among them, 47 alleles, 39 alleles, 41 alleles, and 50 alleles of 51 alleles that can bind to HLA-DR that can be predicted by the high antibody production inducing ability and the HLA-DR restriction prediction program “ProPrep” described later, respectively. MptL (43-62), DiTox (20), TetT1L and TetT3L, which are expected to be able to bind to the allele, are preferred, and DiTox (20) is particularly preferred.

In Silico analysis revealed that DiTox (19), which lacks one amino acid residue at the C-terminus of DiTox (20), has no effect on the antigenicity or multiaggregate function of the peptide. Therefore, since it is predicted that it can bind to the same allele as DiTox (20), it can be preferably used similarly to DiTox (20).

In addition, if necessary, the human peripheral blood to which the peptide vaccine according to the present invention is administered and these T cell epitopes are mixed and cultured, and the rejuvenation reaction is confirmed, or a small amount thereof is administered intradermally. By confirming that delayed hypersensitivity occurs, the presence or absence of memory T cells can be confirmed, and a preferred T cell epitope for a human to be administered can be selected.

As a cell-binding motif of a cell adhesion molecule, the peptide according to the present invention can be retained on the mucosal surface for a long period of time and has an effect of enhancing the ability to induce antibody production by transmucosal administration such as oral and nasal administration Can be used as long as For example, amino acid sequences of other cell binding motifs can be used, including binding motifs for the integrin family. For example, amino acid sequences belonging to the integrin binding motif include RGD, RED, LDV, and PHSRN known as binding motifs present in cell adhesion molecules such as fibronectin, collagen, vitronectin, fibrinogen, laminin, and Tat protein of human immunodeficiency virus. Amino acid sequences such as (SEQ ID NO: 37 in the Sequence Listing), RKK, DGEA (SEQ ID NO: 38 in the Sequence Listing) can be mentioned. In addition, as amino acid sequences of cell binding motifs other than the integrin binding motif, YIGSR (SEQ ID NO: 39 in the sequence listing), IKVAV (SEQ ID NO: 40 in the sequence listing), RFYVVMKK (SEQ ID NO: 41 in the sequence listing), IRVVM (sequence listing) And the like. Among these, peptides consisting of amino acid sequences represented by RGD, RED, and YIGSR are preferable because of their strong ability to induce production of specific antibodies, and RGD is particularly preferable. In the peptide according to the present invention, the amino acid sequence linking site of these cell binding motifs includes the N-terminal side or C-terminal side of the amino acid sequence including the amino acid sequence of the T cell epitope, or the amino acid sequence of the B cell epitope. The amino acid sequence can be selected from a total of four positions on the N-terminal side or C-terminal side, and it may be linked to at least one of them. In particular, the peptide according to the present invention in which the cell binding motif of the cell adhesion molecule is linked to one side or both sides of the N-terminal side or C-terminal side of the amino acid sequence containing the amino acid sequence of the T cell epitope is the amino acid sequence of the B cell epitope. Since the production of an antibody specific for an amino acid sequence containing is particularly enhanced, the antibody linked to the N-terminal side is particularly preferred.

Specific examples of the peptide according to the present invention include a peptide that contains a B cell epitope of Aβ peptide as a site for inducing antibody production and induces antibody production against Aβ. More specifically, RGD-DiTox (19) -VVKK-Aβ (1-15) (SEQ ID NO: 6) represented by the amino acid sequences described in SEQ ID NOS: 6-11, 13, 43 and 44 in the sequence listing, respectively. RGD-DiTox (19) -GKK-Aβ (1-15) (SEQ ID NO: 7), RGD-DiTox (19) -GKK-Aβ (1-13) (SEQ ID NO: 8), RGD-DiTox (19)- GKK-Aβ (1-10) (SEQ ID NO: 9), RGD-DiTox (19) -GKK-Aβ (1-8) (SEQ ID NO: 10), and RGD-DiTox (19) -GKK-Aβ (1-6 ) (SEQ ID NO: 11), RGD-DiTox (19) -VKK-Aβ (1-15) (SEQ ID NO: 13), RGD-DiTox (19) -VKK-Aβ (1-13) (SEQ ID NO: 43), RGD -D An example is iTox (19) -VVKK-Aβ (1-13) (SEQ ID NO: 44).

RGD-DiTox (20) -KK-Aβ (1-13), RGD-DiTox (19) -VKK-Aβ (1-15), RGD-DiTox (19) -VVKK-Aβ (1-15), RGD- DiTox (19) -GKK-Aβ (1-15), RGD-DiTox (19) -GKK-Aβ (1-13), RGD-DiTox (19) -GKK-Aβ (1-10), RGD-DiTox ( 19) -GKK-Aβ (1-8), RGD-DiTox (19) -GKK-Aβ (1-6), Aβ (1-42), RGD-DiTox (19) -VKK-Aβ (1-13) The amino acid sequences of RGD-DiTox (19) -VVKK-Aβ (1-13) and RGD-DiTox (Rev) -KK-Aβ (1-13) and the SEQ ID NOs in the sequence listing are shown in FIG.

The method for producing the peptide according to the present invention is not limited, and can be prepared by a conventional peptide synthesis method, or by combining peptides partially synthesized in advance by a conventional peptide synthesis method. . Moreover, the said peptide can be synthesize | combined according to the protocol of an apparatus using the peptide synthesizer marketed from each manufacturer.

In addition, the peptide according to the present invention can be prepared by recombinant DNA technology. For example, DNA encoding the amino acid sequence of the designed peptide is prepared, inserted into a vector capable of autonomous propagation, and then inserted into a microorganism such as Escherichia coli, Bacillus subtilis, actinomycetes, yeast, animals and plants, or cells or tissues thereof. The peptide according to the present invention can be collected and purified by an appropriate method after it is introduced into the above host and used as a transformant, or transgenic animals and plants are prepared and cultured and grown. Furthermore, the fungus body, animal body or plant body in which the peptide according to the present invention is expressed can be processed as it is and used as a composition for oral consumption containing the peptide according to the present invention. Examples of plant species include Asteraceae, Brassicaceae, Cucurbitaceae, Apiaceae, Rosaceae, Vitaceae, Azalea, and Vaccinium. Family (Caricaceae), legume (Fabaceae), walnut family (Juglandaceae), red crustaceae (Chenopodiaceae), solanaceae (Solanaceae), convolvuleae, cereals (Poaceae) Plant species, more specifically lettuce, chicory, mugwort, broccoli, cabbage, radish, horseradish, mustard, cucumber, melon Pumpkin, chayote, carrot, honeybee, celery, apple, plum, ume, peach, strawberry, raspberry, almond, pear, loquat, grape, cranberry, cowberry, blueberry, papaya, alfalfa, soybean, walnut, spinach, tomato, capsicum, Examples include sweet potato, rice, corn, wheat, barley, rye, yam, and potato. In addition, the peptides according to the present invention may be prepared by directly preparing a peptide having the entire amino acid sequence by any of the methods described above, or by chemically synthesizing peptides having a partial amino acid sequence in advance. It can also be prepared by combining them.

2. Evaluation animal The antibody-producing ability of the peptide vaccine of the present invention can be evaluated using an animal that has established an immunological memory for a peptide containing a T-cell epitope of the peptide vaccine and produces an antibody using a B-cell epitope.

Examples of animal species include mice, dogs, cats, goats, sheep, pigs, and non-human primates (eg, rhesus monkeys, cynomolgus monkeys, chimpanzees).

In order to examine the antibody-producing ability of peptide vaccines against Aβ, for example, RGD peptide, diphtheria toxoid and / or T cell epitope of tetanus toxoid, and B against Aβ were administered to mice preliminarily immunized with diphtheria toxoid and / or tetanus toxoid. A peptide vaccine consisting of cellular epitopes, in which T-cell epitopes and B-cell epitopes are bound by either GKK, VKK, or VVKK linkers, and examining antibody titers in blood and Aβ accumulation in the brain Can be confirmed. The mouse used in the experiment is not limited as long as the effect of the peptide vaccine of the present invention can be confirmed. For example, JU mouse, Bl6 mouse, Balb / c mouse (Nippon Charles River Co., Ltd.) Aβ and Swedish mutation (Swedish mutation) Tg2576 mice (Science, 1996, 274, pp. 99-102) expressing the APP695 isoform containing the K670N and M671L mutations and having enhanced Aβ accumulation ability can be used. .

3. Pharmaceutical Composition As long as the effects of the present invention are not hindered, a pharmaceutical composition in which one or two or more pharmaceutically acceptable excipients are combined with the peptide of the present invention can be prepared. Additives for the preparation include water, alcohol and other solvents, reducing sugars such as glucose and maltose, non-reducing sugars such as α, α-trehalose, sucrose and cyclodextrin, or glucosyl trehalose and malto Carbohydrate derivatives of α, α-trehalose such as syltrehalose, sugar alcohols such as sorbitol, mannitol, maltitol, maltotriitol, water soluble polymers such as agar, pullulan, guar gum, gum arabic, proteins such as gelatin and silk And hydrolysates thereof, lipids, amino acids, buffers, stabilizers, antibacterial agents, fragrances, nutritional functional foods, quasi-drugs or active pharmaceutical ingredients, immune adjuvants such as alum, aluminum hydroxide, and other than the above Food additives, pharmaceutical additives, etc. It can be used in combination of two or more as appropriate. Of these, α, α-trehalose and α, α-trehalose saccharide derivatives, which have a high peptide stabilizing effect, are preferred.

The peptide according to the present invention includes all pharmacologically acceptable salts, esters, or salts of such esters of the peptide. Pharmacologically acceptable salts are preferably alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, iron salt and zinc salt. , Copper salts, nickel salts, metal salts such as cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N- Methyl glucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethyl Ammonium salt, Tris Amine salts such as organic salts such as hydroxymethyl) aminomethane salts; hydrohalogenated hydrohalates such as hydrofluorates, hydrochlorides, hydrobromides, hydroiodates, nitrates, perchlorates Inorganic acid salts such as acid salts, sulfates and phosphates; lower alkane sulfonates such as methane sulfonate, trifluoromethane sulfonate and ethane sulfonate, benzene sulfonate and p-toluene sulfonate Aryl sulfonates, such as aryl sulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, and other organic acid salts; and glycine salt, Amino acid salts such as lysine salts, arginine salts, ornithine salts, glutamates, aspartates can be mentioned.

Further, the form of the preparation containing the peptide according to the present invention is not particularly limited as long as the peptide in the preparation can be stably maintained for a long period of time, and a solution, a lyophilized product, a tablet, a sublingual tablet, What is necessary is just to select suitably from dosage forms, such as a troche, powder, granule, cream, ointment, and syrup, in consideration of an administration subject, an administration method, the preservation | save method of a formulation, and a transportation method. In addition, the peptide according to the present invention or a composition containing the peptide can be encapsulated in liposomes or used in combination with a penetration enhancer or iontophoresis to skin or tissue, if necessary. It is also possible to promote penetration into the existing site. Moreover, the peptide which concerns on this invention can also be transmucosally administered by making it contain in various food-drinks, such as tablet confectionery, a candy, and a soft drink, and ingesting this orally. In addition, the peptide according to the present invention can be expressed in vivo by administering RNA encoding the peptide according to the present invention directly into a living body or by so-called gene therapy in which DNA is introduced into a cell.

The method for administering the pharmaceutical composition containing the peptide of the present invention as an active ingredient to a human is not particularly limited, and any method can be used as long as the peptide of the present invention can reliably reach the administration site. For example, an appropriate amount may be dropped on the mucous membrane using a dropper or syringe, orally taken, applied to the mucosa in the form of a cream or gel, or guided to the administration site with a catheter, etc. Alternatively, it may be sprayed in the form of a mist with a spray or nebulizer, or sucked into the nose, trachea or lung. For administration into body cavities such as subcutaneous, intradermal, intramuscular, intravascular, intraperitoneal and intrathoracic, administration methods such as syringes, catheters and infusions can be used. The dose of the peptide according to the present invention may be appropriately determined in consideration of the antibody production inducing ability, the type of disease, the administration route, the administration method, the animal to be administered, etc., and is generally 0.00001 to 100 mg / kg body weight, It is preferably 0.0001 to 25 mg / kg body weight, more preferably 0.001 to 10 mg / kg body weight. In addition, since the peptide according to the present invention can effectively induce antibody production by the first administration, the subsequent administration is performed only with a peptide having the amino acid sequence of the B cell epitope of this peptide. However, production of the target antibody can be enhanced.

The pharmaceutical composition containing the peptide of the present invention as an active ingredient can be used simultaneously or sequentially with one or more other pharmaceutical compositions. After administering another pharmaceutical composition, a pharmaceutical composition containing the peptide of the present invention as an active ingredient may be administered, or after administering the pharmaceutical composition, another pharmaceutical composition may be administered. In addition, the pharmaceutical composition and the chemotherapeutic agent may be administered simultaneously. Other pharmaceutical compositions vary depending on the target disease, but examples include other vaccines, chemotherapeutic agents, antibody drugs, antisense nucleic acid drugs, siRNA drugs, and the like.

The peptide according to the present invention or a pharmaceutical composition containing the peptide as an active ingredient can be used as a peptide vaccine. A vaccine refers to a formulation administered for the purpose of conferring active immunity against a pathogen. Active immunity refers to inducing an immune response by administration of an antigen. Peptide vaccine refers to a preparation containing a peptide consisting of an amino acid sequence of a part that actually induces an immune response and serves to protect the body of the amino acid sequence of a protein.

The peptide vaccine according to the present invention selects a preferable antigen peptide according to the intended use of the antibody to be produced thereby, thereby preventing infection, inflammatory diseases such as cancer, tumor, ulcer, hepatitis, allergy and atopic skin. It can be used for prevention and treatment of immune diseases such as inflammation. Alternatively, it can be used for the production of antibodies used for detection of various antigens used for neutralization of enzymes, clinical tests, and the like.

In the peptide vaccine according to the present invention, a peptide vaccine that induces the production of anti-Aβ antibody by selecting an amino acid sequence containing an amino acid sequence of an Aβ-derived B cell epitope as an amino acid sequence containing the amino acid sequence of a B cell epitope Obtainable. As such a peptide vaccine, for example, RGD-DiTox (19) -VVKK-Aβ (1-15) represented by the amino acid sequences described in SEQ ID NOs: 6-11, 13, 43 and 44 in the sequence listing, respectively ( SEQ ID NO: 6), RGD-DiTox (19) -GKK-Aβ (1-15) (SEQ ID NO: 7), RGD-DiTox (19) -GKK-Aβ (1-13) (SEQ ID NO: 8), RGD-DiTox (19) -GKK-Aβ (1-10) (SEQ ID NO: 9), RGD-DiTox (19) -GKK-Aβ (1-8) (SEQ ID NO: 10), and RGD-DiTox (19) -GKK-Aβ (1-6) (SEQ ID NO: 11), RGD-DiTox (19) -VKK-Aβ (1-15) (SEQ ID NO: 13), RGD-DiTox (19) -VKK-Aβ (1-13) (SEQ ID NO: 43) and RGD-DiTox (19) -VVKK-Aβ (1-13) (SEQ ID NO: 44), and pharmacologically acceptable salts of the peptide.

The peptide vaccine according to the present invention can be used alone or in combination with one or more other pharmaceuticals. Examples of pharmaceuticals used in combination include acetylcholinesterase inhibitors, NMDA (N-methyl-D-aspartate) glutamate receptor antagonists, anti-Aβ vaccines, anti-Aβ antibodies, γ-secretase inhibitors, β-secretase inhibitors And Aβ aggregation inhibitors. Examples of acetylcholinesterase inhibitors include donepezil hydrochloride, rivastigmine tartrate and galantamine hydrobromide. Specific examples of NMDA (N-methyl-D-aspartate) type glutamate receptor antagonists include memantine hydrochloride. Specific examples of anti-Aβ antibodies include bapinezumab, solanezumab, ponezumab, and gantenrumumab.

The peptide vaccine that induces the production of anti-Aβ antibodies according to the present invention can prevent or treat diseases caused by Aβ. The disease caused by Aβ as used in the present invention refers to a disease caused by Aβ depositing on a specific tissue or the like, and it goes without saying that the deposition is a major cause of the etiology, and Aβ deposition associated with other diseases. Including diseases that develop or exacerbate due to the disease, and also include clinical symptoms associated with these diseases. Specifically, for example, Alzheimer's disease (AD), Alzheimer-type senile dementia (SDAT), senile dementia, frontotemporal dementia, Pick's disease, Down's syndrome, hereditary amyloid cerebral hemorrhage (Dutch type), mild cognition Disorder, memory impairment / learning disorder, amyloidosis, cerebral ischemia, cerebrovascular dementia, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Drager syndrome) , Amyotrophic lateral sclerosis, degenerative ataxia, cortical basal degeneration, Guam ALS-Parkinson-demented complications, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathy, primary progressive aphasia, Motor neuron diseases including striatal substantia nigra degeneration, Machado-Joseph disease / spinal cerebellar ataxia type 3 and olive bridge cerebellar degeneration, Jill de la Tu Tott's disease, bulbar and pseudobulbar paralysis, spinal cord and spinal medullary atrophy (Genedy's disease), primary lateral sclerosis, familial spastic paraplegia, Wernig-Hoffmann disease, Kugelberg-Welander disease, Tay-Sachs Disease, Zandhoff disease, familial spastic disease, Voorfert-Kuegelberg-Welander disease, spastic paraplegia, progressive multifocal leukoencephalopathy, and prion disease (Kreuzfeld-Jakob disease), Gerstmann-Streisler-Scheinker Age-related dementia, vascular dementia, diffuse white matter disease (Winnswanger disease), dementia of endocrine or metabolic origin, head trauma and diffuse brain, including illness, kuru and fatal familial insomnia Infection including dementia of injury, dementia fighting posture or frontal lobe dementia, cerebral ischemia or embolic occlusion and thrombotic occlusion and optional Neurodegenerative diseases resulting from different types of intracranial hemorrhage, intracranial and vertebral damage, hereditary cerebral vascular disorders, non-neuropathic hereditary amyloid, Down's syndrome, macroglobulinemia, secondary familial Mediterranean fever, Maccle- Diabetes due to wellness syndrome, multiple myeloma, amyloidosis associated with pancreatic disease or heart disease, chronic hemodialysis arthritis or Finnish and Iowa amyloidosis, neurodegenerative diseases such as diabetic neuropathy, and Aβ deposition in the pancreas Including clinical symptoms associated with these diseases. Among the above diseases, Alzheimer's disease, Alzheimer type senile dementia, mild cognitive impairment, senile dementia, Down's syndrome or amyloidosis is preferred.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples shown below.

Various Aβ peptide vaccines were prepared and their anti-Aβ antibody production inducing ability was evaluated using Balb / c mice.

The peptide converts the linker site and B cell epitope site of RGD-DiTox (20) -KK-Aβ (1-13) (SEQ ID NO: 12 in the sequence listing), which is a peptide prototype Aβ peptide vaccine developed in Patent Document 2. And synthesized by outsourcing (Operon Biotechnology Co., Ltd.). All peptides were dissolved in PBS to 1 mg / mL.

The peptides evaluated were RGD-DiTox (20) -KK-Aβ (1-13) (SEQ ID NO: 12 in the sequence listing), RGD-DiTox (19) -VKK-Aβ (1-15) (SEQ ID NO: in the sequence listing) 13), RGD-DiTox (19) -VVKK-Aβ (1-15) (SEQ ID NO: 6 in the sequence listing), RGD-DiTox (19) -GKK-Aβ (1-15) (SEQ ID NO: 7 in the sequence listing) RGD-DiTox (19) -GKK-Aβ (1-13) (SEQ ID NO: 8 in the sequence listing), RGD-DiTox (19) -GKK-Aβ (1-10) (SEQ ID NO: 9 in the sequence listing), RGD -DiTox (19) -GKK-Aβ (1-8) (SEQ ID NO: 10 in the sequence listing) and RGD-DiTox (19) -GKK-Aβ (1-6) (arrangement in the sequence listing) There are eight types of column numbers 11).

The peptide was administered to mice as follows. First, 5 weeks old Balb / c mice (Charles River Japan Co., Ltd.) were preliminarily immunized with 50 μL / mouse of precipitated diphtheria tetanus mixed toxoid (manufactured by Kitasato Institute) subcutaneously in the back, and two weeks after immunization. Peptide administration was performed. For administration, 50 μL / mouse (50 μg / mouse) of a peptide solution prepared to 1 mg / mL was subcutaneously administered to the back. One group (N) consisted of 4 animals, and after the initial administration, 6 additional administrations were performed at 2-week intervals. One week after peptide administration, blood was collected from the tail of the mouse to collect plasma components. Protease inhibitor cocktail (Roche Diagnostics Co., Ltd.) was added to plasma to prepare a sample for anti-Aβ antibody measurement. As a negative control group, PBS was subcutaneously administered to the back at 50 μL / mouse.

The amount of anti-Aβ antibody in blood was measured by ELISA using a plate coated with Aβ (1-42) synthetic peptide (manufactured by Anaspec). Specifically, Aβ (1-42) synthetic peptide dissolved in dimethyl sulfoxide (DMSO) was diluted in distilled water, added to a 96-well plate (100 ng / well), and coated overnight at 4 ° C. (overnight). did. The sample was blocked with 1% Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.), the sample for measurement was added at 100 μL, and incubated at 4 ° C. overnight. After completion of the reaction, the plate was washed, horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody (anti-mouse Ig-HRP, manufactured by Amersham) diluted 2000 times was added and incubated at 4 ° C. for 2 hours. After completion of the reaction, the plate was washed with PBS, a chromogenic substrate ABTS (2,2′-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid, manufactured by KPL) was added, and the mixture was incubated at room temperature for 30 minutes. Absorbance at 405 nm was measured. The anti-Aβ antibody was quantified by using an anti-Aβ monoclonal antibody and quantifying the anti-Aβ antibody from a standard curve.

８Eight kinds of evaluated peptides all induced the production of anti-Aβ antibody, and the amount of antibody increased by additional administration (Fig. 1). Among them, RGD-DiTox (19) -GKK-Aβ (1-15) having a GKK linker peptide has the highest anti-Aβ antibody production inducing ability, and RGD which is a conventional peptide after 6 additional administrations It was revealed that anti-Aβ antibody was produced 6 times more than DiTox (20) -KK-Aβ (1-13) (FIG. 1 and Table 1). Other peptides having the GKK linker peptide also showed high anti-Aβ antibody production induction ability (FIG. 1 and Table 1). RGD-DiTox (19) -VVKK-Aβ (1-15) having a VVKK linker peptide also showed high anti-Aβ antibody production inducing ability (FIG. 1 and Table 1). RGD-DiTox (19) -VKK-Aβ (1-15) has the same ability to induce anti-Aβ antibody production as RGD-DiTox (20) -KK-Aβ (1-13) (FIG. 1 and Table) 1). On the other hand, no clear tendency could be found for the B cell epitope (FIG. 1).

From the above results, when the amino acid sequence of the linker peptide is changed to the amino acid sequence represented by GKK, VKK or VVKK, higher anti-Aβ antibody production inducing ability can be obtained than when the amino acid sequence represented by KK is used. It became clear. In addition, the Aβ peptide vaccine in which the amino acid sequence of the linker peptide is an amino acid sequence represented by GKK, VKK or VVKK significantly enhanced antibody production by additional administration. In particular, RGD-DiTox (19) -GKK-Aβ (1-15) has a high anti-Aβ antibody production inducing ability, and was considered useful as a peptide vaccine for the treatment of Alzheimer's disease.

After 6 additional doses were completed, a 1-month withdrawal period was given, and changes in blood anti-Aβ antibody levels due to withdrawal were observed. When a peptide having a linker peptide consisting of an amino acid sequence represented by any one of GKK, VKK, and VVKK was administered, the amount of anti-Aβ antibody in the blood decreased rapidly after the completion of the additional administration. On the other hand, when the conventional peptide RGD-DiTox (20) -KK-Aβ (1-13) was administered, the blood anti-Aβ antibody amount did not decrease and was maintained for 1 month.

Various Aβ peptide vaccines were prepared and their anti-Aβ antibody production inducing ability was evaluated using JU mice. JU Mouse (Quon D. et al., “Formation of beta-amyloid protein deposits in brains of transgenic rice”, Nature, 1991, vol. 352, p. 239-241). used.

The peptide converts the linker site and B cell epitope site of RGD-DiTox (20) -KK-Aβ (1-13) (SEQ ID NO: 12 in the sequence listing), which is a peptide prototype Aβ peptide vaccine developed in Patent Document 2. And synthesized by outsourcing (Operon Biotechnology Co., Ltd.). All peptides were dissolved in PBS to 1 mg / mL.

The peptides evaluated were RGD-DiTox (20) -KK-Aβ (1-13) (SEQ ID NO: 12 in the sequence listing), RGD-DiTox (19) -VKK-Aβ (1-15) (SEQ ID NO: in the sequence listing) 13), RGD-DiTox (19) -VVKK-Aβ (1-15) (SEQ ID NO: 6 in the sequence listing), RGD-DiTox (19) -GKK-Aβ (1-15) (SEQ ID NO: 7 in the sequence listing) , RGD-DiTox (19) -GKK-Aβ (1-13) (SEQ ID NO: 8 in the Sequence Listing).

Administration of various peptides to mice was performed as follows. First, precipitated diphtheria tetanus mixed toxoid (manufactured by Kitasato Institute) was subcutaneously administered to the back of the 8 week-old JU mouse at 50 μL / mouse, preliminary immunization was performed, and peptide administration was performed 2 weeks after the immunization. The peptide was prepared to 200 μg / mL, mixed with an equal amount of adjuvant (Freund Incomplete Adjuvant (FIA), Wako Pure Chemical Industries, Ltd.) to prepare an emulsion, and 100 μg / mouse (10 μg) was subcutaneously applied to the back of JU mice. / Mouse). One group (N) was used as 4 animals, and after the first administration, two additional administrations were performed at 2-week intervals. One week after the second additional administration, blood was collected from the tail of the mouse to collect plasma components. Protease inhibitor cocktail (Roche Diagnostics Co., Ltd.) was added to plasma to prepare a sample for anti-Aβ antibody measurement. As a negative control group, PBS and an equal amount of FIA were mixed to prepare an emulsion, and 100 μL / mouse was subcutaneously administered to the back.

The blood anti-Aβ antibody amount was measured according to the same method as the anti-Aβ antibody amount measuring method described in Example 1.

All of the five kinds of peptides evaluated induced anti-Aβ antibody production by two additional administrations (Table 2). Among them, RGD-DiTox (19) -GKK-Aβ (1-15) having a GKK linker peptide has the highest ability to induce anti-Aβ antibody production, and RGD-DiTox (20) -KK, which is a conventional peptide. Induced 85-fold production of anti-Aβ antibody compared to -Aβ (1-13) (Table 2). Similarly, RGD-DiTox (19) -GKK-Aβ (1-13) and RGD-DiTox (19) -VVKK-Aβ (1-15) having a GKK linker peptide are also RGD-DiTox (20) -KK. As compared with -Aβ (1-13), the anti-Aβ antibody production inducing ability was more than 10 times higher (Table 2). In the administration by emulsion, the same results as in the peptide solution administration could be confirmed.

From the above results, even in the case of administration using adjuvant, the amino acid sequence of the linker peptide is changed to the amino acid sequence represented by GKK, VKK or VVKK, which is higher than the amino acid sequence represented by KK. It was revealed that anti-Aβ antibody production induction ability was obtained. In particular, RGD-DiTox (19) -GKK-Aβ (1-15) has a high anti-Aβ antibody production inducing ability, and was considered useful as a peptide vaccine for the treatment of Alzheimer's disease.

Various Aβ peptide vaccines were prepared and their anti-Aβ antibody production inducing ability was evaluated using Balb / c mice.

Peptides RGD-DiTox (20) -KK-Aβ (1-13) (SEQ ID NO: 12 in the sequence listing), RGD-DiTox (19) -VKK-Aβ (1-13) (SEQ ID NO: 43 in the sequence listing), and Three types of RGD-DiTox (19) -VVKK-Aβ (1-13) (SEQ ID NO: 44 in the sequence listing) were synthesized and dissolved in PBS to a concentration of 1 mg / mL.

The peptide was administered to mice as follows. First, 6 weeks old Balb / c mice (Charles River Japan Co., Ltd.) were preliminarily immunized with 50 μL / mouse of the precipitated diphtheria tetanus mixed toxoid (Kitasato Laboratory) subcutaneously in the back, and two weeks after immunization. Peptide administration was performed. For administration, 50 μL / mouse (50 μg / mouse) of a peptide solution prepared to 1 mg / mL was intraperitoneally administered. 1 group (N) 4-6 animals (4 negative control group, RGD-DiTox (19) -VVKK-Aβ (1-13) group 6 animals, other peptide group 5 animals), 2 weeks after initial administration Six additional doses were given at intervals. One week after peptide administration, blood was collected from the tail of the mouse to collect plasma components. Protease inhibitor cocktail (Roche Diagnostics Co., Ltd.) was added to plasma to prepare a sample for anti-Aβ antibody measurement. As a negative control group, PBS was intraperitoneally administered at 50 μL / mouse.

The amount of anti-Aβ antibody in blood was measured by ELISA using a plate coated with Aβ (1-42) synthetic peptide (manufactured by Anaspec). Specifically, Aβ (1-42) synthetic peptide dissolved in dimethyl sulfoxide (DMSO) was diluted in distilled water, added to a 96-well plate (100 ng / well), and coated overnight at 4 ° C. (overnight). did. The sample was blocked with 1% Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.), the sample for measurement was added at 100 μL, and incubated at 4 ° C. overnight. After completion of the reaction, the plate was washed, horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody (anti-mouse Ig-HRP, manufactured by Amersham) diluted 2000 times was added and incubated at 4 ° C. for 2 hours. After completion of the reaction, the plate was washed with PBS, a chromogenic substrate ABTS (2,2′-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid, manufactured by KPL) was added, and the mixture was incubated at room temperature for 30 minutes. Absorbance at 405 nm was measured. The anti-Aβ antibody was quantified by using an anti-Aβ monoclonal antibody and quantifying the anti-Aβ antibody from a standard curve.

All of the three types of peptides evaluated induced the production of anti-Aβ antibody, and the amount of antibody increased by additional administration (FIG. 2). Among them, RGD-DiTox (19) -VVKK-Aβ (1-13) having a VVKK linker peptide is a conventional peptide RGD-DiTox (20) -KK-Aβ (1-13) after 6 additional administrations. It was revealed that three times as many anti-Aβ antibodies were produced (FIG. 2 and Table 3). The ability of RGD-DiTox (19) -VKK-Aβ (1-13) having VKK linker peptide after 6 additional administrations to induce anti-Aβ antibody production is RGD-DiTox (20) -KK-Aβ (1-13) (Fig. 2 and Table 3).

From the above results, it is clear that by using the amino acid sequence of the linker peptide as the amino acid sequence represented by VVKK, it is possible to obtain higher anti-Aβ antibody production inducing ability than using the amino acid sequence represented by KK. became.

Various Aβ peptide vaccines were prepared, and their anti-Aβ antibody production inducing ability was evaluated using JU-Tg2576 mice. JU-Tg2576 is a mouse obtained by backcrossing a Tg2576 mouse and a JU mouse. JU-Tg2576 mice were used after self-breeding.

RGD-DiTox (20) -KK-Aβ (1-13) (SEQ ID NO: 12 in the sequence listing), RGD-DiTox (Rev) -KK-Aβ (1-13) (sequence) with the T cell epitope site reversed (SEQ ID NO: 45 in the sequence table), RGD-DiTox (19) -VKK-Aβ (1-15) (SEQ ID NO: 13 in the sequence listing), RGD-DiTox (19) -VVKK-Aβ (1-13) (in the sequence listing) SEQ ID NO: 44) and RGD-DiTox (19) -VVKK-Aβ (1-15) (SEQ ID NO: 6 in the sequence listing) were each dissolved in PBS so as to be 1 mg / ml.

Administration of various peptides to mice was performed as follows. First, JU-Tg2576 mice aged 3 months were preliminarily immunized with 50 μL / mouse of precipitated diphtheria tetanus mixed toxoid (Kitasato Laboratory) subcutaneously in the back, and the peptide was administered 2 weeks after the immunization. The peptide is prepared to 1 mg / mL, mixed with an equal amount of adjuvant (Freund Incomplete Adjuvant (FIA), manufactured by Wako Pure Chemical Industries, Ltd.) to prepare an emulsion, and 100 μL / mouse is intraperitoneally in a JU-Tg2576 mouse. (50 μg / mouse). 1 group (N) 9-20 (20 negative control group, RGD-DiTox (20) -KK-Aβ (1-13) group 17 mice, RGD-DiTox (19) -VVKK-Aβ (1-13) 11 animals in the group, 10 animals in the RGD-DiTox (19) -VKK-Aβ (1-15) group and RGD-DiTox (19) -VVKK-Aβ (1-15) group, RGD-DiTox (Rev) -KK-Aβ (1-13) group 9 animals), and after the first administration, 6 additional administrations were performed at 2-week intervals. One week after peptide administration, blood was collected from the tail of the mouse to collect plasma components. Protease inhibitor cocktail (Roche Diagnostics Co., Ltd.) was added to plasma to prepare a sample for anti-Aβ antibody measurement. Further, as a negative control group, PBS and an equal amount of FIA were mixed to prepare an emulsion, which was intraperitoneally administered at 100 μL / mouse.

The amount of anti-Aβ antibody in blood was measured by ELISA using a plate coated with Aβ (1-42) synthetic peptide (manufactured by Anaspec). Specifically, Aβ (1-42) synthetic peptide dissolved in dimethyl sulfoxide (DMSO) was diluted in distilled water, added to a 96-well plate (100 ng / well), and coated overnight at 4 ° C. (overnight). did. The sample was blocked with 1% Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.), the sample for measurement was added at 100 μL, and incubated at 4 ° C. overnight. After completion of the reaction, the plate was washed, horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody (anti-mouse Ig-HRP, manufactured by Amersham) diluted 2000 times was added and incubated at 4 ° C. for 2 hours. After completion of the reaction, the plate was washed with PBS, a chromogenic substrate ABTS (2,2′-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid, manufactured by KPL) was added, and the mixture was incubated at room temperature for 30 minutes. Absorbance at 405 nm was measured. The anti-Aβ antibody was quantified by using an anti-Aβ monoclonal antibody and quantifying the anti-Aβ antibody from a standard curve.

In RGD-DiTox (Rev) -KK-Aβ (1-13) with the T cell epitope site reversed, no anti-Aβ antibody production was induced (FIG. 3). The other four peptides all induced the production of anti-Aβ antibody, and the amount of antibody increased by additional administration (FIG. 3). Among them, RGD-DiTox (19) -VKK-Aβ (1-15) having a VKK linker peptide continues until after 6 additional administrations, and RGD-DiTox (20) -KK-Aβ (conventional peptide) continues. It was revealed that anti-Aβ antibody was produced higher than in 1-13) (FIG. 3 and Table 4). Other peptides having the VVKK linker peptide also showed high anti-Aβ antibody production induction ability (FIG. 3 and Table 4).

From the above results, it can be seen that, when the amino acid sequence of the linker peptide is changed to the amino acid sequence represented by VKK or VVKK, higher anti-Aβ antibody production inducing ability can be obtained than when the amino acid sequence represented by KK is used. It became clear. In addition, the Aβ peptide vaccine in which the amino acid sequence of the linker peptide is an amino acid sequence represented by VKK or VVKK significantly enhanced antibody production by additional administration.

As described above, the present invention relates to an amino acid sequence (T) that includes an amino acid sequence of a T cell epitope that has established immunological memory in a living body, and an amino acid that includes an amino acid sequence of one or more specific B cell epitopes of an antigen. Comprising a sequence (B) and an amino acid sequence of a linker peptide arranged to link them between T and B, wherein the amino acid sequence of the linker peptide is GKK, VKK or VVKK A peptide and a composition containing the peptide as an active ingredient are provided. For example, the present invention provides a peptide having an anti-Aβ antibody production-inducing ability having such a structure and a pharmaceutical composition containing the peptide as an active ingredient. Since the peptides according to the present invention are restricted by a wide range of human HLA-DR haplotypes, they are caused by foreign substances such as pathogens and viruses in many humans, particularly in humans who have established an immunological memory by vaccination. It can be used as a peptide vaccine effective for prevention and treatment of various diseases. Alternatively, it can be used as a peptide vaccine that induces antibody production against Aβ, which is effective in the prevention and treatment of various diseases such as neurodegenerative diseases including Alzheimer's disease. Further, the peptide vaccine according to the present invention exhibits the ability to induce antibody production without using an immunoadjuvant, and can be used for transmucosal administration such as nasal administration or oral administration. It is a simple and safe peptide vaccine because the medium concentration decreases rapidly.

SEQ ID NO: 1: Amyloid β peptide 1-42.
SEQ ID NO: 2: Amyloid β peptide 1-40.
Sequence number 3: Synthetic peptide.
SEQ ID NO: 4: Peptide consisting of amino acid residues 1-15 of amyloid β peptide
SEQ ID NO: 5: Peptide consisting of amino acid residues 1-13 of amyloid β peptide
SEQ ID NO: 6: Synthetic peptide, referred to herein as RGD-DiTox (19) -VVKK-Aβ (1-15).
SEQ ID NO: 7: synthetic peptide, designated herein as RGD-DiTox (19) -GKK-Aβ (1-15)
SEQ ID NO: 8: synthetic peptide, designated herein as RGD-DiTox (19) -GKK-Aβ (1-13)
SEQ ID NO: 9: synthetic peptide, designated herein as RGD-DiTox (19) -GKK-Aβ (1-10)
SEQ ID NO: 10: synthetic peptide, designated herein as RGD-DiTox (19) -GKK-Aβ (1-8)
SEQ ID NO: 11: synthetic peptide, designated herein as RGD-DiTox (19) -GKK-Aβ (1-6)
SEQ ID NO: 12: synthetic peptide, designated herein as RGD-DiTox (20) -KK-Aβ (1-13)
SEQ ID NO: 13: synthetic peptide, referred to herein as RGD-DiTox (19) -VKK-Aβ (1-15)
SEQ ID NO: 14: peptide consisting of amino acid residues 1-11 of amyloid β peptide
SEQ ID NO: 15: peptide consisting of amino acid residues 3-11 of amyloid β peptide
SEQ ID NO: 16: peptide consisting of amino acid residues 3-15 of amyloid β peptide
SEQ ID NO: 17: Peptide consisting of amino acid residues 1-10 of amyloid β peptide
SEQ ID NO: 18: peptide consisting of amino acid residues 1-8 of amyloid β peptide
SEQ ID NO: 19: peptide consisting of amino acid residues 1-6 of amyloid β peptide
SEQ ID NO: 20: Partial peptide of diphtheria toxoid.
SEQ ID NO: 21: Partial peptide of diphtheria toxoid.
SEQ ID NO: 22: Partial peptide of diphtheria toxoid.
SEQ ID NO: 23: Partial peptide of tetanus toxoid.
SEQ ID NO: 24: Partial peptide of tetanus toxoid.
SEQ ID NO: 25: Partial peptide of tetanus toxoid.
SEQ ID NO: 26: Partial peptide of tetanus toxoid.
SEQ ID NO: 27: Partial peptide of tetanus toxoid.
SEQ ID NO: 28: Partial peptide of tetanus toxoid.
SEQ ID NO: 29: Partial peptide of tetanus toxoid.
SEQ ID NO: 30: partial peptide of tetanus toxoid.
SEQ ID NO: 31: partial peptide of tetanus toxoid.
SEQ ID NO: 32: partial peptide of tetanus toxoid.
SEQ ID NO: 33: Partial peptide of secreted protein MPT64 of Mycobacterium bovine
SEQ ID NO: 34: Partial peptide of the secretory protein MPT64 of Mycobacterium tuberculosis
SEQ ID NO: 35: Partial peptide of secretory protein MPT64 of Mycobacterium tuberculosis
SEQ ID NO: 36: Partial peptide of secretory protein MPT64 of Mycobacterium tuberculosis
SEQ ID NO: 37: peptide containing cell adhesion motif
SEQ ID NO: 38: Peptide containing cell adhesion motif
SEQ ID NO: 39: peptide containing cell adhesion motif
SEQ ID NO: 40: peptide containing cell adhesion motif
SEQ ID NO: 41: peptide containing cell adhesion motif
SEQ ID NO: 42: peptide containing cell adhesion motif
SEQ ID NO: 43: synthetic peptide, designated herein as RGD-DiTox (19) -VKK-Aβ (1-13)
SEQ ID NO: 44: synthetic peptide, designated herein as RGD-DiTox (19) -VVKK-Aβ (1-13)
SEQ ID NO: 45: synthetic peptide, designated herein as RGD-DiTox (Rev) -KK-Aβ (1-13)

Claims (22)

1. A peptide having an amino acid sequence represented by the following general formula or a salt thereof, wherein T represents an amino acid sequence including the amino acid sequence of a T cell epitope for which immunological memory is established, L represents an amino acid sequence of a linker peptide, B Is an amino acid sequence comprising the amino acid sequence of one or more specific B cell epitopes of the antigen, wherein at least one of R1, R2, R3 and R4 represents a peptide binding amino acid sequence of a cell adhesion molecule or A peptide or a salt thereof, characterized in that the amino acid sequence of the linker peptide is an amino acid sequence represented by GKK, VKK or VVKK.
   (Chemical formula 1)
   R1-T-R2-L-R3-B-R4
2. 2. The T cell epitope has an amino acid sequence derived from any one or two or more peptides selected from peptides used as antigens for vaccination of diphtheria, tuberculosis, tetanus, and pertussis. Or a salt thereof.
3. The peptide or a salt thereof according to claim 1 or 2, wherein the B cell epitope has an amino acid sequence including the amino acid sequence of the B cell epitope of amyloid β peptide.
4. The amino acid sequence containing the amino acid sequence of the B cell epitope of amyloid β peptide is the amino acid sequence from the first amino acid to the 15th amino acid sequence (SEQ ID NO: 4 in the sequence listing) from the N terminus of amyloid β or the amino acid sequence from the N terminus of amyloid β. The peptide or salt thereof according to claim 3, which is the amino acid sequence from the first amino acid to the 13th amino acid sequence (SEQ ID NO: 5 in the sequence listing).
5. The peptide or a salt thereof according to claim 4, comprising an amino acid sequence selected from SEQ ID NOs: 6 to 11, 13, 43 and 44 in the sequence listing.
6. A peptide comprising an amino acid sequence selected from SEQ ID NOs: 6 to 11, 13, 43 and 44 in the sequence listing, or a salt thereof.
7. The peptide which consists of an amino acid sequence of sequence number 6 in a sequence table, or its salt.
8. The peptide which consists of an amino acid sequence of sequence number 7 in a sequence table, or its salt.
9. The peptide which consists of an amino acid sequence of sequence number 8 in a sequence table, or its salt.
10. The peptide which consists of an amino acid sequence of sequence number 9 in a sequence table, or its salt.
11. The peptide which consists of an amino acid sequence of sequence number 10 in a sequence table, or its salt.
12. The peptide which consists of an amino acid sequence of sequence number 11 in a sequence table, or its salt.
13. The peptide which consists of an amino acid sequence of sequence number 13 in a sequence table, or its salt.
14. The peptide which consists of an amino acid sequence of sequence number 43 in a sequence table, or its salt.
15. The peptide which consists of an amino acid sequence of sequence number 44 in a sequence table, or its salt.
16. A pharmaceutical composition comprising as an active ingredient at least one peptide selected from claims 1 to 15 or a pharmacologically acceptable salt thereof.
17. The pharmaceutical composition according to claim 16, further comprising one or more pharmaceutical additives that are pharmaceutically acceptable.
18. Inducing antibody production against Aβ containing one or more pharmaceutically acceptable excipients together with the peptide according to any one of claims 3 to 15 or a pharmacologically acceptable salt thereof. A pharmaceutical composition capable of
19. The pharmaceutical composition according to any one of claims 16 to 18, which is used for prevention and / or treatment of a disease caused by Aβ.
20. The use according to any one of claims 16 to 18, which is used for the prevention and / or treatment of at least one disease selected from Alzheimer's disease, Alzheimer-type senile dementia, mild cognitive impairment, senile dementia, Down's syndrome and amyloidosis. The pharmaceutical composition according to item.
21. The pharmaceutical composition according to any one of claims 16 to 18, which is used for prevention and / or treatment of Alzheimer's disease.
22. DNA encoding the peptide according to any one of claims 1 to 15.

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