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WO2011090065A1 - Vaccin peptidique - Google Patents

Vaccin peptidique Download PDF

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Publication number
WO2011090065A1
WO2011090065A1 PCT/JP2011/050848 JP2011050848W WO2011090065A1 WO 2011090065 A1 WO2011090065 A1 WO 2011090065A1 JP 2011050848 W JP2011050848 W JP 2011050848W WO 2011090065 A1 WO2011090065 A1 WO 2011090065A1
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WIPO (PCT)
Prior art keywords
peptide
amino acid
acid sequence
sequence
salt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2011/050848
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English (en)
Japanese (ja)
Inventor
佳人 金澤
充裕 牧野
尚克 三輪
明 矢野
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hayashibara Seibutsu Kagaku Kenkyujo KK
Daiichi Sankyo Co Ltd
Original Assignee
Hayashibara Seibutsu Kagaku Kenkyujo KK
Daiichi Sankyo Co Ltd
Hayashibara Biochemical Laboratories Co Ltd
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Application filed by Hayashibara Seibutsu Kagaku Kenkyujo KK, Daiichi Sankyo Co Ltd, Hayashibara Biochemical Laboratories Co Ltd filed Critical Hayashibara Seibutsu Kagaku Kenkyujo KK
Publication of WO2011090065A1 publication Critical patent/WO2011090065A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • 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 ⁇ peptide (hereinafter abbreviated as “A ⁇ peptide”), and A ⁇ containing the peptide as an active ingredient.
  • a ⁇ peptide human amyloid ⁇ peptide
  • 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.
  • 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.
  • Non-patent Document 1 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).
  • Non-patent Document 1 Is known as an immune adjuvant
  • 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.
  • 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”).
  • 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.
  • 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.
  • the polypeptide has an immune adjuvant effect.
  • 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 (
  • T amino acid sequence
  • B an amino acid sequence that includes one or more B cell epitope amino acid sequences of the A ⁇ peptide on the C-terminal side, in some cases, abbreviated as “L”.
  • 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.
  • AD Alzheimer's disease
  • QOL quality of life
  • 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 A ⁇ accumulates in the central nerve, neurofibrillary tangles, and neuronal degeneration and loss.
  • cholinesterase inhibitors and anti-hyperlipidemic agents and cranial nerve protective agents such as ethyl eicosapentaenoic acid (EPA) are being conducted.
  • EPA ethyl eicosapentaenoic acid
  • Non-patent Document 2 Development of new adjuvants such as human anti-A ⁇ monoclonal antibodies, DNA vaccines, CpGDNA, and peptide vaccines for oral administration is also underway.
  • 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.
  • peptide vaccines are weakly immunogenic and have a low ability to induce antibody production against A ⁇ , an adjuvant is often required to induce antibody production, and an excessive immune reaction may be caused by the use of an adjuvant.
  • 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 A ⁇ 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.
  • a peptide that efficiently induces the production of antibodies against A ⁇ 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 A ⁇ 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).
  • Patent Literature 2).
  • 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 A ⁇ 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.
  • the present invention relates to: 1.
  • 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
  • at least one of R1, R2, R3 and R4 is cell adhesion
  • 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.
  • the B cell epitope has an amino acid sequence including the amino acid sequence of the B cell epitope of A ⁇ peptide. Or 2. Peptide or salt thereof, 4).
  • the amino acid sequence including the amino acid sequence of the B cell epitope of the A ⁇ 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 A ⁇ , or the first amino acid sequence from the N terminus of A ⁇ . 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 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 capable of inducing production of an antibody against A ⁇ 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 A ⁇ .
  • At least one disease selected from Alzheimer's disease, Alzheimer-type senile dementia, mild cognitive impairment, senile dementia, Down's syndrome and amyloidosis.
  • For the prevention and / or treatment of Alzheimer's disease To 18. Any pharmaceutical composition of 22. 1 above.
  • 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.
  • 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.
  • the present invention 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 A ⁇ can be a great advantage because it has few such side effects.
  • RGD-DiTox (19) -GKK-A ⁇ 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-A ⁇ antibody production, and increased production of anti-A ⁇ antibody was observed even after 6 additional administrations.
  • VVKK linker peptide Other peptides having the GKK linker peptide also showed high anti-A ⁇ antibody production inducing ability.
  • Veh indicates the result of administration of phosphate buffered saline (PBS) instead of the A ⁇ peptide vaccine.
  • PBS phosphate buffered saline
  • 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.
  • “Vehicle” indicates the result of administration of phosphate buffered saline (PBS) instead of the A ⁇ peptide vaccine.
  • “KK1-13” represents RGD-DiTox (20) -KK-A ⁇ (1-13).
  • VKK1-13 indicates RGD-DiTox (19) -VKK-A ⁇ (1-13).
  • VVKK1-13 indicates RGD-DiTox (19) -VVKK-A ⁇ (1-13).
  • Example 3 It is a figure which shows the result of having evaluated the anti-A ⁇ antibody production induction ability after the additional administration of various A ⁇ peptide vaccines using JU-Tg2576 mice.
  • “Vehicle” indicates the result of administration of phosphate buffered saline (PBS) instead of the A ⁇ peptide vaccine.
  • Tcell rev indicates RGD-DiTox (Rev) -KK-A ⁇ (1-13).
  • KK1-13 represents RGD-DiTox (20) -KK-A ⁇ (1-13).
  • VKK1-15 represents RGD-DiTox (19) -VKK-A ⁇ (1-15).
  • VVKK1-13 indicates RGD-DiTox (19) -VVKK-A ⁇ (1-13).
  • VVKK1-15 indicates RGD-DiTox (19) -VVKK-A ⁇ (1-15).
  • Example 4 It is a figure which shows the amino acid sequence and sequence number of various peptides.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • an A ⁇ peptide vaccine prepared using a dipeptide composed of KK as a linker peptide sometimes did not increase the amount of anti-A ⁇ antibody produced when repeated boosting was performed, and the degree of increase was low.
  • an A ⁇ peptide vaccine prepared using a peptide consisting of an amino acid sequence represented by GKK, VKK or VVKK as a linker peptide is an A ⁇ peptide vaccine prepared using a dipeptide consisting of KK as a linker peptide, as shown in the Examples described later.
  • the anti-A ⁇ antibody production inducing ability was higher, and the increase in the amount of antibody produced by additional administration was also large.
  • 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 A ⁇ peptide. it can.
  • the A ⁇ 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 A ⁇ peptide vaccine prepared using a dipeptide consisting of KK as a linker peptide.
  • the medium anti-A ⁇ antibody concentration decreases rapidly.
  • 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.
  • a vaccine against an internal antigen such as A ⁇ has a great advantage that it has few side effects.
  • the rapid reduction of the blood anti-A ⁇ antibody concentration after the end of administration of the A ⁇ peptide vaccine prepared using a peptide consisting of the amino acid sequence represented by GKK, VKK or VVKK as a linker peptide includes an A ⁇ -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 A ⁇ peptide, the blood concentration of the target antibody can be quickly reduced after the administration is completed.
  • the A ⁇ 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 A ⁇ 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.
  • 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.
  • 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.
  • 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.
  • the processing of each epitope can be performed more reliably.
  • a peptide that induces antibody production against A ⁇ can be produced by using the B cell epitope of A ⁇ peptide as a site that induces antibody production.
  • the “B cell epitope of A ⁇ peptide” refers to a peptide comprising the amino acid sequence of the B cell epitope of A ⁇ that causes Alzheimer's disease.
  • a peptide having an amino acid sequence that can dissolve A ⁇ aggregation fibers, inhibit the deposition of A ⁇ into nerve fibers and other organs, and inhibit the binding to acetylcholine receptor is preferable.
  • a peptide including an amino acid sequence near the N-terminus of A ⁇ 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 A ⁇ may be linked in tandem.
  • the entire A ⁇ for example, 42 amino acids (SEQ ID NO: 1 in the sequence listing, hereinafter “A ⁇ ( 1-42) ”or 40 (SEQ ID NO: 2 in the sequence listing, hereinafter abbreviated as“ A ⁇ (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.
  • the amino acid sequence near the N-terminus of A ⁇ when used as the B cell epitope, it preferably has at least the 4-10th amino acid sequence from the N-terminus of A ⁇ .
  • 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.
  • a ⁇ (1-15) 1-13th A peptide having the amino acid sequence of DAEFRHDSGYEVH (SEQ ID NO: 5 in the Sequence Listing, hereinafter abbreviated as “A ⁇ (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 “A ⁇ (1-11)”.) 3-1.
  • Peptide having the first amino acid sequence of EFRHDSGYE (SEQ ID NO: 15 in the Sequence Listing, hereinafter abbreviated as “A ⁇ (3-11)”), Peptide having the amino acid sequence of 3-15th EFRHDSGYEVHHQ (in the Sequence Listing) SEQ ID NO: 16, hereinafter abbreviated as “A ⁇ (3-15)”).
  • a ⁇ (1-10) 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 “A ⁇ (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“ A ⁇ (1-8)
  • 1-6th DAEFFRH SEQ ID NO: 19 in the sequence listing, hereinafter abbreviated as “A ⁇ (1-6)”.
  • a ⁇ (1-15) or A ⁇ (1-13) is preferably used, and A ⁇ (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.
  • 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.
  • 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.
  • HLA-DR molecule which is an MHC class II molecule, and its aggregope
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 LIHVVLHG
  • 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 (S
  • 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).
  • 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.
  • 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.
  • 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
  • transmucosal administration such as oral and nasal administration
  • amino acid sequences of other cell binding motifs can be used, including binding motifs for the integrin family.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • peptide according to the present invention include a peptide that contains a B cell epitope of A ⁇ peptide as a site for inducing antibody production and induces antibody production against A ⁇ . More specifically, RGD-DiTox (19) -VVKK-A ⁇ (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-A ⁇ (1-15) (SEQ ID NO: 7), RGD-DiTox (19) -GKK-A ⁇ (1-13) (SEQ ID NO: 8), RGD-DiTox (19)- GKK-A ⁇ (1-10) (SEQ ID NO: 9), RGD-DiTox (19) -GKK-A ⁇ (1-8) (SEQ ID NO: 10), and RGD-DiTox (19) -GKK-A ⁇ (1-6 ) (SEQ ID NO: 11), RGD-DiTox (19) -VKK-A ⁇ (1-15) (SEQ ID NO: 13), RGD-DiTox (19) -VKK-A ⁇ (1-13) (SEQ ID NO: 43), RGD -D An example is iTox (19) -VVKK-A ⁇ (1-13) (SEQ ID NO: 44).
  • the amino acid sequences of RGD-DiTox (19) -VVKK-A ⁇ (1-13) and RGD-DiTox (Rev) -KK-A ⁇ (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
  • the peptide according to the present invention can be prepared by recombinant DNA technology.
  • 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.
  • 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.
  • plant species include Asteraceae, Brassicaceae, Cucurbitaceae, Apiaceae, Rosaceae, Vitaceae, Azalea, and Vaccinium.
  • 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.
  • 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.
  • 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.
  • mice examples include mice, dogs, cats, goats, sheep, pigs, and non-human primates (eg, rhesus monkeys, cynomolgus monkeys, chimpanzees).
  • non-human primates eg, rhesus monkeys, cynomolgus monkeys, chimpanzees.
  • peptide vaccines against A ⁇ for example, RGD peptide, diphtheria toxoid and / or T cell epitope of tetanus toxoid, and B against A ⁇ 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 A ⁇ 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.
  • JU mouse, Bl6 mouse, Balb / c mouse (Nippon Charles River Co., Ltd.) A ⁇ 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 A ⁇ accumulation ability can be used. .
  • 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 ⁇ , ⁇ -trehalose, sucrose and cyclodextrin, or glucosyl trehalose and malto Carbohydrate derivatives of ⁇ , ⁇ -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
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • it may be sprayed in the form of a mist with a spray or nebulizer, or sucked into the nose, trachea or lung.
  • 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.
  • 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.
  • 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.
  • a peptide vaccine that induces the production of anti-A ⁇ antibody by selecting an amino acid sequence containing an amino acid sequence of an A ⁇ -derived B cell epitope as an amino acid sequence containing the amino acid sequence of a B cell epitope Obtainable.
  • RGD-DiTox (19) -VVKK-A ⁇ (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-A ⁇ (1-15) (SEQ ID NO: 7), RGD-DiTox (19) -GKK-A ⁇ (1-13) (SEQ ID NO: 8), RGD-DiTox (19) -GKK-A ⁇ (1-10) (SEQ ID NO: 9), RGD-DiTox (19) -GKK-A ⁇ (1-8) (SEQ ID NO: 10), and RGD-DiTox (19) -GKK-A ⁇ (1-6) (SEQ ID NO: 11), RGD-DiTox (19) -VKK-A ⁇ (1-15) (SEQ ID NO: 13), RGD-DiTox (19) -VKK-A ⁇ (1-13) (SEQ ID NO: 6), and RGD-DiTox
  • the peptide vaccine according to the present invention can be used alone or in combination with one or more other pharmaceuticals.
  • pharmaceuticals used in combination include acetylcholinesterase inhibitors, NMDA (N-methyl-D-aspartate) glutamate receptor antagonists, anti-A ⁇ vaccines, anti-A ⁇ antibodies, ⁇ -secretase inhibitors, ⁇ -secretase inhibitors And A ⁇ aggregation inhibitors.
  • 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.
  • anti-A ⁇ antibodies include bapinezumab, solanezumab, ponezumab, and gantenrumumab.
  • the peptide vaccine that induces the production of anti-A ⁇ antibodies according to the present invention can prevent or treat diseases caused by A ⁇ .
  • the disease caused by A ⁇ as used in the present invention refers to a disease caused by A ⁇ depositing on a specific tissue or the like, and it goes without saying that the deposition is a major cause of the etiology, and A ⁇ deposition associated with other diseases. Including diseases that develop or exacerbate due to the disease, and also include clinical symptoms associated with these diseases.
  • AD Alzheimer's disease
  • SDAT Alzheimer-type senile dementia
  • 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 Step-Richardson syndrome
  • Multisystem degeneration Shoy-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
  • the peptide converts the linker site and B cell epitope site of RGD-DiTox (20) -KK-A ⁇ (1-13) (SEQ ID NO: 12 in the sequence listing), which is a peptide prototype A ⁇ 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-A ⁇ (1-13) (SEQ ID NO: 12 in the sequence listing), RGD-DiTox (19) -VKK-A ⁇ (1-15) (SEQ ID NO: in the sequence listing) 13), RGD-DiTox (19) -VVKK-A ⁇ (1-15) (SEQ ID NO: 6 in the sequence listing), RGD-DiTox (19) -GKK-A ⁇ (1-15) (SEQ ID NO: 7 in the sequence listing) RGD-DiTox (19) -GKK-A ⁇ (1-13) (SEQ ID NO: 8 in the sequence listing), RGD-DiTox (19) -GKK-A ⁇ (1-10) (SEQ ID NO: 9 in the sequence listing), RGD -DiTox (19) -GKK-A ⁇ (1-8) (SEQ ID NO: 10 in the sequence listing) and RGD-DiTox (19) -GKK-A ⁇ (1-6) (arrangement in the sequence listing
  • mice 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 ⁇ L / 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 ⁇ L / mouse (50 ⁇ g / 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.
  • the amount of anti-A ⁇ antibody in blood was measured by ELISA using a plate coated with A ⁇ (1-42) synthetic peptide (manufactured by Anaspec). Specifically, A ⁇ (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 ⁇ L, and incubated at 4 ° C. overnight.
  • DMSO dimethyl sulfoxide
  • 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.
  • 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-A ⁇ antibody was quantified by using an anti-A ⁇ monoclonal antibody and quantifying the anti-A ⁇ antibody from a standard curve.
  • RGD-DiTox (19) -VVKK-A ⁇ (1-15) having a VVKK linker peptide also showed high anti-A ⁇ antibody production inducing ability (FIG. 1 and Table 1).
  • RGD-DiTox (19) -VKK-A ⁇ (1-15) has the same ability to induce anti-A ⁇ antibody production as RGD-DiTox (20) -KK-A ⁇ (1-13) (FIG. 1 and Table) 1).
  • no clear tendency could be found for the B cell epitope (FIG. 1).
  • the amino acid sequence of the linker peptide is changed to the amino acid sequence represented by GKK, VKK or VVKK, higher anti-A ⁇ antibody production inducing ability can be obtained than when the amino acid sequence represented by KK is used. It became clear.
  • the A ⁇ 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.
  • RGD-DiTox (19) -GKK-A ⁇ (1-15) has a high anti-A ⁇ antibody production inducing ability, and was considered useful as a peptide vaccine for the treatment of Alzheimer's disease.
  • JU Mouse Quality 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-A ⁇ (1-13) (SEQ ID NO: 12 in the sequence listing), which is a peptide prototype A ⁇ 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-A ⁇ (1-13) (SEQ ID NO: 12 in the sequence listing), RGD-DiTox (19) -VKK-A ⁇ (1-15) (SEQ ID NO: in the sequence listing) 13), RGD-DiTox (19) -VVKK-A ⁇ (1-15) (SEQ ID NO: 6 in the sequence listing), RGD-DiTox (19) -GKK-A ⁇ (1-15) (SEQ ID NO: 7 in the sequence listing) , RGD-DiTox (19) -GKK-A ⁇ (1-13) (SEQ ID NO: 8 in the Sequence Listing).
  • mice Animals administered to mice were 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 ⁇ L / mouse, preliminary immunization was performed, and peptide administration was performed 2 weeks after the immunization.
  • the peptide was prepared to 200 ⁇ g / mL, mixed with an equal amount of adjuvant (Freund Incomplete Adjuvant (FIA), Wako Pure Chemical Industries, Ltd.) to prepare an emulsion, and 100 ⁇ g / mouse (10 ⁇ g) 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-A ⁇ antibody measurement.
  • PBS and an equal amount of FIA were mixed to prepare an emulsion, and 100 ⁇ L / mouse was subcutaneously administered to the back.
  • the blood anti-A ⁇ antibody amount was measured according to the same method as the anti-A ⁇ antibody amount measuring method described in Example 1.
  • RGD-DiTox (20) -KK-A ⁇ (1-13) SEQ ID NO: 12 in the sequence listing
  • RGD-DiTox (19) -VKK-A ⁇ (1-13) SEQ ID NO: 43 in the sequence listing
  • Three types of RGD-DiTox (19) -VVKK-A ⁇ (1-13) SEQ ID NO: 44 in the sequence listing were synthesized and dissolved in PBS to a concentration of 1 mg / mL.
  • mice 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 ⁇ L / 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 ⁇ L / mouse (50 ⁇ g / mouse) of a peptide solution prepared to 1 mg / mL was intraperitoneally administered.
  • N 1 group (N) 4-6 animals (4 negative control group, RGD-DiTox (19) -VVKK-A ⁇ (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-A ⁇ antibody measurement.
  • PBS was intraperitoneally administered at 50 ⁇ L / mouse.
  • the amount of anti-A ⁇ antibody in blood was measured by ELISA using a plate coated with A ⁇ (1-42) synthetic peptide (manufactured by Anaspec). Specifically, A ⁇ (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 ⁇ L, and incubated at 4 ° C. overnight.
  • DMSO dimethyl sulfoxide
  • 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.
  • 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-A ⁇ antibody was quantified by using an anti-A ⁇ monoclonal antibody and quantifying the anti-A ⁇ antibody from a standard curve.
  • RGD-DiTox (19) -VVKK-A ⁇ (1-13) having a VVKK linker peptide is a conventional peptide RGD-DiTox (20) -KK-A ⁇ (1-13) after 6 additional administrations. It was revealed that three times as many anti-A ⁇ antibodies were produced (FIG. 2 and Table 3).
  • the ability of RGD-DiTox (19) -VKK-A ⁇ (1-13) having VKK linker peptide after 6 additional administrations to induce anti-A ⁇ antibody production is RGD-DiTox (20) -KK-A ⁇ (1-13) (Fig. 2 and Table 3).
  • JU-Tg2576 mice is a mouse obtained by backcrossing a Tg2576 mouse and a JU mouse. JU-Tg2576 mice were used after self-breeding.
  • mice fed various peptides to mice as follows.
  • JU-Tg2576 mice aged 3 months were preliminarily immunized with 50 ⁇ L / 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 ⁇ L / mouse is intraperitoneally in a JU-Tg2576 mouse. (50 ⁇ g / mouse).
  • FFA Fluorincomplete Adjuvant
  • N 9-20 (20 negative control group, RGD-DiTox (20) -KK-A ⁇ (1-13) group 17 mice, RGD-DiTox (19) -VVKK-A ⁇ (1-13) 11 animals in the group, 10 animals in the RGD-DiTox (19) -VKK-A ⁇ (1-15) group and RGD-DiTox (19) -VVKK-A ⁇ (1-15) group, RGD-DiTox (Rev) -KK-A ⁇ (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-A ⁇ 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 ⁇ L / mouse.
  • the amount of anti-A ⁇ antibody in blood was measured by ELISA using a plate coated with A ⁇ (1-42) synthetic peptide (manufactured by Anaspec). Specifically, A ⁇ (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 ⁇ L, and incubated at 4 ° C. overnight.
  • DMSO dimethyl sulfoxide
  • 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.
  • 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-A ⁇ antibody was quantified by using an anti-A ⁇ monoclonal antibody and quantifying the anti-A ⁇ antibody from a standard curve.
  • 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.
  • T amino acid sequence
  • B 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.
  • the present invention provides a peptide having an anti-A ⁇ antibody production-inducing ability having such a structure and a pharmaceutical composition containing the peptide as an active ingredient.
  • 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 A ⁇ , 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 ⁇ peptide 1-42.
  • SEQ ID NO: 2 Amyloid ⁇ peptide 1-40.
  • Sequence number 3 Synthetic peptide.
  • SEQ ID NO: 4 Peptide consisting of amino acid residues 1-15 of amyloid ⁇ peptide
  • SEQ ID NO: 5 Peptide consisting of amino acid residues 1-13 of amyloid ⁇ peptide
  • SEQ ID NO: 6 Synthetic peptide, referred to herein as RGD-DiTox (19) -VVKK-A ⁇ (1-15).
  • SEQ ID NO: 7 synthetic peptide, designated herein as RGD-DiTox (19) -GKK-A ⁇ (1-15)
  • SEQ ID NO: 8 synthetic peptide, designated herein as RGD-DiTox (19) -GKK-A ⁇ (1-13)
  • SEQ ID NO: 9 synthetic peptide, designated herein as RGD-DiTox (19) -GKK-A ⁇ (1-10)
  • SEQ ID NO: 10 synthetic peptide, designated herein as RGD-DiTox (19) -GKK-A ⁇ (1-8)
  • SEQ ID NO: 13 synthetic peptide, referred to herein as RGD-DiTox (19) -VKK-A ⁇ (1-15)
  • 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-A ⁇

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Abstract

La présente invention a pour objet un peptide qui possède une bonne immunogénicité et est capable d'induire une productivité accrue d'anticorps et dont une administration supplémentaire peut accroître la production de l'anticorps cible. La présente invention concerne spécifiquement un peptide comprenant la séquence d'acides aminés d'un motif se liant aux cellules d'une molécule d'adhérence cellulaire, une séquence d'acides aminés (T) contenant la séquence d'acides aminés d'un épitope de lymphocyte T ayant une mémoire immunologique établie, une séquence d'acides aminés (B) contenant la ou les séquences d'acides aminés d'un ou plusieurs épitopes de lymphocytes B spécifiques d'un antigène, et la séquence d'acides aminés d'un peptide de liaison qui est positionnée entre les séquences d'acides aminés (T) et (B) susdites de sorte à lier ces séquences d'acides aminés, caractérisé en ce que la séquence d'acides aminés dudit peptide de liaison est GKK, VKK ou VVKK. La présente invention concerne également une composition médicale qui comprend le peptide susdit en tant que principe actif et son utilisation.
PCT/JP2011/050848 2010-01-21 2011-01-19 Vaccin peptidique Ceased WO2011090065A1 (fr)

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JP2010010953A JP2013090574A (ja) 2010-01-21 2010-01-21 ペプチドワクチン
JP2010-010953 2010-01-21

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2015050158A1 (fr) * 2013-10-01 2015-04-09 国立大学法人三重大学 Vaccin d'induction de lymphocytes t contenant une séquence interépitope favorisant une présentation d'antigène
WO2018113023A1 (fr) * 2016-12-23 2018-06-28 四川大学 Nouveau vaccin polypeptidique contre l'asthme et procédé de préparation à cet effet

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2004087767A1 (fr) * 2003-03-31 2004-10-14 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Polypeptide
WO2007097251A1 (fr) * 2006-02-22 2007-08-30 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo VACCIN PEPTIDIQUE DESTINE A INDUIRE LA PRODUCTION D'ANTICORPS ANTI-PEPTIDE AMYLOIDE β

Patent Citations (2)

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WO2004087767A1 (fr) * 2003-03-31 2004-10-14 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Polypeptide
WO2007097251A1 (fr) * 2006-02-22 2007-08-30 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo VACCIN PEPTIDIQUE DESTINE A INDUIRE LA PRODUCTION D'ANTICORPS ANTI-PEPTIDE AMYLOIDE β

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ADAMS, M. J. ET AL.: "Overview and analysis of the polyprotein cleavage sites in the family Potyviridae.", MOL. PLANT PATHOL., vol. 6, no. 4, 2005, pages 471 - 487 *
AKIRA YONO: "Saibo Ketsugo Hairetsu o Mochiita Ikyushusei Peptide Seizai no Sekkei", RESEARCH OF NEW MEDICAL DEVICES, vol. 11, 25 March 2006 (2006-03-25), pages 16, XP003017253 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015050158A1 (fr) * 2013-10-01 2015-04-09 国立大学法人三重大学 Vaccin d'induction de lymphocytes t contenant une séquence interépitope favorisant une présentation d'antigène
US11179450B2 (en) 2013-10-01 2021-11-23 Mie University Long chain antigen containing interepitope sequence that promotes antigen presentation to T cells
WO2018113023A1 (fr) * 2016-12-23 2018-06-28 四川大学 Nouveau vaccin polypeptidique contre l'asthme et procédé de préparation à cet effet

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TW201138815A (en) 2011-11-16

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