HK1130810A - PEPTIDE VACCINE FOR INDUCING PRODUCTION OF ANTI-AMYLOID-β-PEPTIDE ANTIBODY - Google Patents

Description

Peptide vaccine for inducing anti-amyloid beta peptide antibody production

Technical Field

The present invention relates to a peptide having excellent immunogenicity and capable of efficiently inducing the production of antibodies against amyloid β peptide (hereinafter, referred to as "a β"), a composition containing the peptide as an active ingredient, and use thereof. More specifically, the present invention relates to a peptide that is effective for inducing an antibody against a specific epitope of a β on a B cell, and a composition for preventing and/or treating a neuropathy typified by alzheimer's disease, which contains the peptide as an active ingredient.

Background

The dramatic increase in the number of patients with cognitive diseases has become a serious problem in various developed countries where aging progresses rapidly. The cognitive diseases can be classified into cerebrovascular cognitive diseases due to cerebrovascular disorders and alzheimer's disease. Among them, alzheimer's disease is a disease that causes memory impairment or dyskinesia and significantly lowers QOL, which accounts for the majority of the causes of senile cognitive diseases. In addition to the treatment of diseases, a large amount of care is required, and this is a disease that not only causes medical expenses, but also imposes various social burdens such as a large amount of time loss, a loss of labor opportunities, and mental burdens on the patient himself or a caregiver typified by his family. The above-mentioned alzheimer disease is a serious neurodegenerative disease without fundamental treatment, and the pathology thereof is a disease characterized in that a β composed of 40 (seq id No. 1 in the sequence listing) or 42 (seq id No. 2 in the sequence listing) amino acids accumulates in the central nerve and is pigmentation of so-called "senile plaque", neurofibrillary change, and degeneration and loss of nerve cells. In the treatment of alzheimer's disease, there have been carried out a treatment using a cholinesterase inhibitor and a clinical test using a hyperlipemia inhibitor, a cerebral neuroprotective agent such as ethyl EPA, and the like.

In order to radically cure this, development of a vaccine targeting a β, which plays a major role in alzheimer's disease, is also underway (see, for example, Agadjanyan, m.g., Journal of Immunology, volume 174, No. 3, pages 1580 to 1586 (2005)). In addition, development of human anti-a β monoclonal antibodies, DNA vaccines, novel adjuvants such as CpGDNA, and peptide vaccines for oral administration is also underway. However, DNA vaccines have major histocompatibility complex antigen (hereinafter, abbreviated as "MHC") class I restriction and are easy to induce activation of cytotoxic T cells, while peptide vaccines have poor immunogenicity and lack the ability to effectively induce antibodies against a β, and therefore, it is necessary to use an adjuvant for antibody induction, and when antigens are large molecules, T cell and B cell epitopes are often contained, and there is a high possibility that an immune response to the above-mentioned epitopes causes an undesirable inflammatory reaction, and there is a risk of side effects such as meningitis, and therefore, they have not yet been established as a fundamental treatment method for alzheimer's disease. In addition, a β is known to be involved in the onset and progression of various diseases other than alzheimer's disease, but a method for preventing or treating the above-mentioned diseases has not yet been established.

On the other hand, in order to solve the above problems, the present inventors have disclosed an amino acid sequence peptide vaccine having a cell-binding motif on a polypeptide having an amino acid sequence of a T cell epitope on the amino acid terminal side and an amino acid sequence of a B cell epitope on the carbonyl terminal side thereof via a linker peptide, which can induce the production of an antibody against a β peptide and is used for the treatment of alzheimer's disease (see international publication No. WO 2004/87767). However, the specification of International publication WO2004/87767 does not describe a specific amino acid sequence of a Peptide vaccine for solving the problem, and it has been also proved that the ability to induce anti-A β antibodies is weak even when a repetitive, shifted multiple antigen restriction site type Peptide (Overlapping multiepitope Peptide shown in sequence No. 3 of the sequence Listing, hereinafter abbreviated as "OMP") described in the specification of International publication WO2004/87767 or a polypeptide having the sequence of amino acid at position 298-312 of Gag protein (sequence No. 4 of the sequence Listing, hereinafter abbreviated as "Gag") derived from human immunodeficiency virus (hereinafter abbreviated as "HIV") is used as an epitope of T cells. In order to induce anti-a β antibodies in a large number of alzheimer's disease patients as possible using the above-mentioned polypeptides as vaccines, it is necessary to design T cell epitopes that can induce strong anti-a β antibodies, while being restricted by a large number of human MHC types II, that is, haplotypes (alleles) of HLA-DR, and being linked to B cell epitopes of a β. Furthermore, although it is necessary to suppress the development of symptoms of vaccines for the prevention and/or treatment of various neurodegenerative diseases and the like caused by other than a β based on alzheimer's disease at an early stage, it is desirable to have a peptide which induces anti-a β antibodies efficiently with the minimum number of administration times and has a low risk of inducing serious side effects when administered to a human in view of economy.

Disclosure of Invention

Disclosed is a peptide vaccine for preventing and/or treating various diseases such as a neurodegenerative disease typified by Alzheimer's disease caused by A beta.

The present inventors have made intensive studies and experiments to solve the above problems, and as a result, have found that a peptide vaccine having an excellent ability to induce the production of an antibody against a B cell epitope can be produced without using an adjuvant by selecting a T cell epitope contained in a peptide of a sensitizing antigen called an inactivated vaccine or toxin for vaccination, which is administered in advance as a peptide containing a T cell epitope, utilizing the immunological memory already existing in the human body. Further, it has been found that a peptide having a specific site of a β as a B cell epitope, which is bound to a peptide as the T cell epitope and a linker peptide at an interval to form a polypeptide having an amino acid sequence of a cell binding motif of a cell adhesion molecule in the peptide, can effectively induce an antibody recognizing only a specific region part of a β even when administered via a mucosal route such as a nasal cavity or an oral cavity. Furthermore, by selecting a plurality of multi-antigen restriction site type epitopes restricted by HLA-DR as T-cell epitopes, HLA-DR restriction problems can be avoided, and a polypeptide having 1 amino acid sequence can effectively induce antibodies recognizing only a specific region part of A.beta.in a plurality of human individuals. The present invention has been completed based on the above findings.

That is, the present invention provides a polypeptide having an amino acid sequence represented by the following general formula, wherein T represents an amino acid sequence of a T cell epitope for which immunological memory has been established, L is an amino acid sequence of a linker peptide, B represents an amino acid sequence of a B cell epitope containing 1 or 2 or more sites of A β, and R is an amino acid sequence of a B cell epitope for which A β can be induced to be produced efficiently₁、R₂、R₃And R₄At least 1 of which represents an amino acid sequence of a cell binding motif of a cell adhesion factor, and a composition for preventing and/or treating various diseases caused by a β, including neurodegenerative diseases represented by alzheimer's disease, which contains the polypeptide.

A compound of the general formula:

R₁-T-R₂-L-R₃-B-R₄

Detailed Description

The peptide used for inducing an anti-a β antibody in the present invention refers to a peptide having an amino acid sequence represented by the general formula and having an ability to induce production of an antibody against a B cell epitope of a β or an amino acid sequence containing the same.

The antibody of the present invention is mainly an immunoglobulin g (igg), an immunoglobulin m (igm), and an immunoglobulin a (iga) antibody, and is derived from an antibody secreted in blood or body fluid, and naturally includes an antibody secreted in a nasal cavity, an oral cavity, an eye, a digestive tract, and the like.

The diseases caused by a β according to the present invention include diseases caused by a β precipitation in a specific tissue and the like, and include diseases caused or aggravated by a β precipitation accompanying other diseases although the precipitation is not a main cause, and clinical symptoms accompanying the above diseases. Specific examples thereof include, for example, alzheimer's disease, pick's disease; pericardial pseudocirrhosis, diffuse lewy body disease, progressive supranuclear palsy (basal gangliosis syndrome), multiple system atrophy (Shy-Drager syndrome), amyotrophic lateral sclerosis, degenerative ataxia, corticobasal degeneration, mannich ALS-parkinson-dementia complications, subacute sclerosing pan encephalitis, huntington's chorea, parkinson's disease, synucleinopathy, primary progressive aphasia, schlieren degeneration, motor neuron disease including cerebellar degenerative spinal dyskinesia third type and olivopontocerebellar atrophy, twitch dirty syndrome (gilles de la tourette syndrome), bulbar palsy and pseudobulbar palsy, spinal and spinal bulbar muscular atrophy (kennedy syndrome), primary lateral sclerosis, familial spastic paraplegia, wiry-hode syndrome, kuguerbet-welan disease, kugue-warerkin disease, parkinson's disease, creutzfeldt-jakob disease, lejacob disease, cre, Tay-saxophone disease, sandoft's disease, familial spasticity, woo-kupffer-weizi disease, spastic paraplegia, progressive multifocal leukoencephalopathy, prion disease (creutzfeldt-jakob disease), gerstmann-straussler-scheinker disease, age-elevation-related dementia including kuru and fatal familial insomnia, vascular dementia, diffuse leukopathy (bessema), dementia due to endocrine and metabolism, dementia due to head injury or diffuse brain injury, disoriented aggressive posture or frontal lobe dementia, neurodegenerative disease caused by infarction including cerebral ischemia, embolic infarction or thrombotic infarction, and neuropathy caused by any kind of intracranial hemorrhage, intracerebral injury, spinal injury, hereditary cerebral vascular disorder, non-neurological hereditary amyloidosis, cerebral ischemia, down's syndrome, macroglobulinemia, secondary familial mediterranean fever, Mueller-Weldii syndrome, multiple myeloma, amyloidosis associated with pancreatic or cardiac disease, chronic hemodialysis arthropathy, amyloidosis of Finland or Iowa type, neurodegenerative diseases such as diabetic neuropathy, diabetes caused by precipitation of A β in the pancreas, and clinical symptoms associated with the above diseases.

The peptide of the present invention is a polypeptide having an amino acid sequence of a B cell epitope of a β that is a cause of alzheimer's disease as a B cell epitope of a β that is a site for inducing antibody production. Among them, a polypeptide having an amino acid sequence that can dissolve a β aggregated fibers, inhibit the deposition of a β on nerve fibers or other organs, and induce the production of an antibody that can inhibit the binding to acetylcholine is desired, and from this point of view, a polypeptide having an amino acid sequence at the amino terminus of a β, preferably consisting of about 5 to 20 amino acids, is particularly preferred. When the number of constituent amino acids is too small, the antibody-inducing ability is lost, and when too large, the antigenicity is too high, and there is a possibility that the epitope of the antibody may contain an adverse side effect. The peptide containing the B cell epitope may be used as it is, or a 2-mer, 3-mer or multimer thereof may be used in tandem. If necessary, 2 or more types of B cell epitopes present in a β may be connected in series, or a β having, for example, 40 amino acids (seq id No. 1, hereinafter simply referred to as "a β (1-40)") or 42 amino acids (seq id No. 2, hereinafter simply referred to as "a β (1-42)") may be used as a B cell epitope in the whole a β. In addition, by linking a plurality of epitopes, a peptide capable of simultaneously inducing a plurality of antibodies can also be designed at will. In the above case, treatment of each epitope can be more reliably performed by inserting a linker peptide described later between each B cell epitope.

When an amino acid sequence near the amino acid terminus is used as the B cell epitope, it is necessary to have at least the amino acid sequences at positions 4 to 10 from the amino terminal of A.beta., and examples thereof include a peptide having the amino acid at positions 1 to 11 of the amino acid sequences shown in SEQ ID Nos. 1 or 2 of the sequence ID Nos. 1 to 13 (SEQ ID NO: 5, hereinafter abbreviated as "A.beta. (1-11)"), a peptide having the amino acid sequence at positions 1 to 13 (SEQ ID NO: 6, hereinafter abbreviated as "A.beta. (1-13)"), a peptide having the amino acid sequence at positions 1 to 15 (SEQ ID NO: 7, hereinafter abbreviated as "A.beta. (1-15)"), and a peptide having the amino acid sequence at positions 3 to 11 (SEQ ID NO: 8, hereinafter "A.beta. (3-11)") of the amino acid sequences at positions 3 to 11 (SEQ ID NO: EFRHDSGYE of the sequence ID Nos. 7, hereinafter abbreviated as "A., A peptide having an amino acid sequence of EFRHDSGYEVHHQ at positions 3 to 15 (SEQ ID NO: 9 in the sequence listing, hereinafter referred to simply as "A.beta. (3-15)"), and the like. From the viewpoint of the ability to induce anti-A.beta.antibodies or side effects, A.beta. (1-13) or A.beta. (1-15) is preferably used, and A.beta. (1-13) is particularly preferably used.

The T cell epitope that is already present in human immunological memory among the peptides used in the present invention means a T cell epitope that is already present in an administered subject when the peptide vaccine of the present invention is administered, and for example, a peptide containing the same T cell epitope as the peptide of the present invention can be administered in advance to a patient administered with the peptide of the present invention to establish immunological memory. On the other hand, T cell epitopes of antigenic peptides having immunological competence obtained by vaccination from the young children and later in most people can be used without performing the above-described operation for establishing immunological memory. In addition, for a patient whose immunological memory has been established, a better effect can be expected if the above operation is performed again. Since T-cell epitopes are restricted by HLA-DR haplotypes and are recommended as antigens for helper T-cells, it is preferable to use as many repetitive shift type multiple antigen restriction site type peptides or amino acid sequences containing them as possible, which are restricted to HLA-DR haplotypes (alleles). The T cell epitope used in the present invention can preferably use, for example, diphtheria, tetanus, pertussis vaccines, sublingual adenitis, rubella, measles, tuberculosis, and the like as peptides as antigens for preventive vaccination because they establish immunological memory in most people from the infancy, and in particular, vaccination with three mixed vaccines containing tetanus toxoid, diphtheria toxoid, pertussis vaccine, or BCG (tuberculosis), is particularly preferable from the viewpoint of general usability because vaccination is mandatory in japan and is generalized worldwide. Since the amino acid sequence of the above-mentioned peptide is already known, the known amino acid sequence of the T cell epitope of the peptide can be used, and in practice, the epitope sequence of the peptide in the antigen portion can be measured by a cell production reaction in peripheral blood, and the peptide having the measured amino acid sequence can be used as the T cell epitope. Furthermore, a T-cell epitope prediction program for HLA-DR that can be used is employed to select a plurality of multi-antigen restricted epitope regions that bind to HLA-DR and to arbitrarily determine the amino acid sequence of the peptide. In addition, although the T cell epitope itself is preferably low in the ability to induce the production of an antibody against the epitope from the viewpoint of reducing side effects, if a peptide derived from the amino acid sequence of a sensitizing antigen of an epitope for vaccination is used, the epitope is originally used in a human body to induce the production of an antibody against the epitope. Therefore, even if an antibody against the epitope is produced, the safety thereof has been established for a long time, and side effects are unlikely to occur, which is an advantage. The above-mentioned T-cell epitope peptide may be used as it is, or may be used in combination with one another in a tandem manner using the same or a plurality of different types of T-cell epitopes. In addition, in the T cell epitope, an amino acid residue other than the amino acid residue at the restriction site (the amino acid residue that must bind to the HLA-DR antigen) can still function even if it is substituted with another amino acid residue.

Specific examples of the T cell epitope include epitopes having the following amino acid sequences: a peptide derived from the amino acid sequence of AYNFVESIINLFQVVHNSYN which is an epitope of diphtheria endotoxin (SEQ ID NO: 10 in the sequence Listing) and a peptide derived from the amino acid sequence of YNFVESIINLFQVVHNSYN (SEQ ID NO: 11 in the sequence Listing) (hereinafter, each of them is abbreviated as "DTL" or "DTS 19"), a peptide derived from LQTMVKLFNRIKNNVA which is an epitope of tetanus toxoid (SEQ ID NO: 12 in the sequence Listing), a peptide derived from FLQTMVKLFNRIKNNVAG (SEQ ID NO: 13 in the sequence Listing), a peptide derived from IHVLHGLYGMQVSSHE (SEQ ID NO: 14 in the sequence Listing), a peptide derived from LIHVLHGLYGMQVSSHEI (SEQ ID NO: 15 in the sequence Listing), a peptide derived from YIVNEDKFQILYNSIMYG (SEQ ID NO: 16 in the sequence Listing), a peptide derived from QYIVNEDKFQILYNSIMYGF (SEQ ID NO: 17 in the sequence Listing), a peptide derived from SYQMYRSLEYQVDAI (SEQ ID NO: 18 in the sequence Listing), a peptide derived from RSYQMYRSLEYQVDAI (SEQ ID NO: 19 in the sequence Listing), and, A peptide having an amino acid sequence of NINIFMRESSRSFLV (SEQ ID NO: 20 in the sequence Listing), a peptide having an amino acid sequence of ININIFMRESSRSFLVN (SEQ ID NO: 21 in the sequence Listing) (hereinafter, abbreviated as "TetT 1", "TetT 1L", "TetT 2", "TetT 2L", "TetT 3", "TetT 3L", "TetT 4", "TetT 4L", "TetT 5", "TetT 5L", respectively); an amino acid sequence such as a peptide having an amino acid sequence of IQMSDPAYNINISLPSYYPD derived from MPT64 (hereinafter referred to simply as "MPT") known as a protein co-secreted by Mycobacterium tuberculosis (hereinafter referred to simply as "MPT"), a peptide having an amino acid sequence of IQMSDPAYNINISLPS (sequence No. 23 in the sequence Listing), a peptide having an amino acid sequence of DPAYNINISLPSYYPD (sequence No. 24 in the sequence Listing), and a peptide having an amino acid sequence of YNINISLPSYYPDQKS (sequence No. 25 in the sequence Listing) (hereinafter referred to simply as "MptL (43-62)", "MptN (43-58)", "MptM (47-62)", "MptC (50-65)", respectively). Among them, it is desirable to use MptL (43-62), DTL, TetT1L, and TetT3L, preferably DTL, capable of binding to 47 allele, 39 allele, 41 allele, OR 50 allele among 51 alleles capable of binding to HLA-OR by the HLA-OR restriction prediction program "proprep" described later, because of its high ability to induce antibody production. Furthermore, the T cell epitope to be administered to a human may be selected arbitrarily by culturing the peripheral blood of a human to which the vaccine peptide of the present invention is administered in a mixed state with the T cell epitope to confirm the presence or absence of a juvenile reaction, or by administering a small amount of the vaccine peptide into the skin to confirm the presence or absence of delayed hypersensitivity (DTH) and the presence or absence of memory T cells.

The linker peptide used in the present invention for linking a peptide containing a B cell epitope and a T cell epitope may be a recognition sequence of a protease involved in antigen production, and specifically, there may be mentioned dipeptides such as lysine-lysine (KK), lysine-arginine (KR), and arginine-arginine (RR), and among them, a dipeptide comprising lysine-lysine as a recognition sequence of cathepsin B is preferably used.

The cell-binding motif of the cell adhesion molecule used in the present invention is not particularly limited as long as the peptide of the present invention can be retained on the mucosal surface for a long period of time, and can be used in the present invention as long as it has an effect of enhancing the antibody production-inducing ability by mucosal administration such as oral or nasal administration. For example, amino acid sequences of other cell binding motifs, as represented by binding motifs for the integrin family, can also be used. For example, the amino acid sequence belonging to the integrin binding motif can be a peptide having the following amino acid sequence, namely, arginine-glycine-aspartic acid (hereinafter abbreviated as "RGD"), arginine-glutamic acid-aspartic acid (hereinafter abbreviated as "RED"), leucine-aspartic acid-valine (hereinafter abbreviated as "LDV"), proline-histidine-serine-arginine-asparagine (PHSRN: SEQ ID NO: 26 in the sequence Listing), arginine-lysine (RKK), aspartic acid-glycine-glutamic acid-alanine (DGEA: SEQ ID NO: 27 in the sequence Listing), which are known as binding motifs present on cell adhesion molecules such as fibronectin-binding protein, collagen, vitronectin, fibrinogen, laminin, Tat protein of HIV, and the like. In addition, as binding motifs outside the integrin family, there can be mentioned peptides consisting of the following amino acid sequences: tyrosine-isoleucine-glycine-serine-arginine (YIGSR: SEQ ID NO: 28 in the sequence Listing), isoleucine-lysine-valine-alanine-valine (IKVAV: SEQ ID NO: 29 in the sequence Listing), arginine-phenylalanine-tyrosine-valine-methionine-tryptophan-lysine (RFYVVMWK: SEQ ID NO: 30 in the sequence Listing), isoleucine-arginine-valine-methionine (IRVVM: SEQ ID NO: 31 in the sequence Listing), and the like. Among these, peptides composed of the amino acid sequences of RGD, RED, and YIGSR are preferable because they have a strong ability to induce the production of specific antibodies, and RGD is particularly preferable. The linking site of the peptide having the amino acid sequence of the cell binding motif may be selected from 4 sites in total on the amino terminus or the carboxyl terminus side of the peptide of the T cell epitope or on the amino terminus or the carboxyl terminus side of the peptide of the cell epitope, and may be linked to at least 1 site thereof. That is, the peptide of the present invention in which the cell binding motif of the cell adhesion molecule is linked to the amino terminal side or the carboxyl terminal side of the peptide of the T cell epitope or to both sides thereof is preferable, and the peptide linked to the amino terminal side is particularly preferable, because the production of a peptide-specific antibody to the B cell epitope is particularly enhanced.

The method for producing the peptide of the present invention is not particularly limited, and the peptide can be produced by a conventional peptide synthesis method or by binding a partially synthesized peptide in advance by a conventional peptide synthesis method. The peptide was synthesized using a peptide synthesizer commercially available from each manufacturer according to the instructions of the apparatus. In addition, the peptides of the present invention can be prepared by recombinant DNA techniques. For example, the following procedure may be used: DNA encoding the amino acid sequence of the designed peptide is prepared, inserted into a vector capable of autonomous replication, introduced into microorganisms such as Escherichia coli, Bacillus subtilis, Actinomycetes, and yeasts, animals and plants, or hosts such as cells or tissues thereof to prepare transformants, and the transformants are prepared into transgenic animals and plants, cultured and cultured, and then the peptide of the present invention is extracted and purified by an appropriate method. In addition, when the peptide of the present invention is used by selecting an appropriate amino acid sequence of a protease cleavage site for ligation and expression, but not an amino acid sequence of a relevant cleavage site for ligation of a T-cell epitope and a B-cell epitope, the protease cleavage product can be freely selected and used. In addition, the cell, animal or plant expressing the present invention may be directly processed and used as a composition for oral ingestion containing the peptide of the present invention. Examples of the plant include plants of species of the family Asteraceae (Asteraceae), Cruciferae (Brassicaceae), Cucurbitaceae (Cucurbitaceae), Umbelliferae (Apiaceae), Rosaceae (Rosaceae), Vitaceae (Vitaceae), Vaccinium (Vaccinium), Caricaceae (Caricaceae), Leguminosae (Fabaceae), Juglandaceae (Juglandaceae), Chenopodiaceae (Chenopodiaceae), Solanaceae (Solanaceae), Convolvulaceae (Convolvulaceae), Poaceae (Poaceae), Dioscoreaceae (Dioscoreaceae), Convolvulaceae (Convolvulaceae), and the like; more specifically, lettuce, chicory, chrysanthemum, broccoli, cabbage, radish, horseradish, pepper, cucumber, melon, pumpkin, chayote, carrot, hornwort, celery, apple, plum, apricot, peach, strawberry, raspberry, almond, pear, loquat, grape, cranberry, raspberry, blueberry, papaya, alfalfa, soybean, walnut, spinach, tomato, red pepper, sweet potato, rice, maize, wheat, barley, rye, dioscorea japonica, potato, and the like can be cited. The peptide of the present invention can be produced by any of the above-mentioned methods, by directly producing a peptide having a complete amino acid sequence or by chemically binding peptides having an amino acid sequence of a part thereof by synthesis to each other, and is not particularly limited.

The peptide of the present invention may be appropriately prepared as a composition by using 1 or 2 or more pharmaceutically acceptable additives in combination with the peptide of the present invention within a range not to impair the effects of the present invention. Examples of the additives for pharmaceutical preparations include solvents such as water and ethanol, and reducing sugars such as glucose, maltose, trehalose, and sucrose; alpha, alpha-trehalose derivatives such as alpha, alpha-trehalose and alpha-maltosylmaltose, cyclic tetrasaccharides having a structure of ring { → 6) -alpha-D-glucopyranosyl- (1 → 3) -alpha-D-glucopyranosyl- (1 → 6) disclosed in the specification of International publication No. WO 02/10361, and ring { → 6) -alpha-D-glucopyranosyl- (1 → 4) disclosed in Japanese unexamined patent publication No. 2005-A Non-reducing sugars such as cyclic tetrasaccharides and cyclodextrins, sugar alcohols such as sorbitol, mannitol, maltitol, and maltotriose; water-soluble polymers such as agar, pullulan, guar gum, and gum arabic; proteins such as gelatin and silk, or hydrolysates thereof; lipids, amino acids, buffers, stabilizers, antibacterial agents, perfumes, effective components in nutritional functional foods, pharmaceutical cosmetics and medicines, immunoadjuvants such as alum and aluminum hydroxide, food additives other than the above substances, pharmaceutical additives, etc., and optionally 1 or more than 2 of the above substances may be used in combination. Among these, α -trehalose, saccharide derivatives of α, α -trehalose, and cyclic tetrasaccharides having a high peptide stabilizing effect are preferable.

The form of the preparation containing the peptide of the present invention is not particularly limited as long as the peptide in the preparation can be stably stored for a long period of time, and may be appropriately selected in consideration of the administration subject, the administration method, the method of storing the preparation, or the method of transporting the preparation, from the dosage forms such as a solution, a lyophilized product, a tablet, a sublingual tablet, a buccal preparation, an ointment, and a syrup. The peptide of the present invention or a composition containing the same can be encapsulated in a liposome, or used in combination with a skin or tissue penetration enhancer or an iontophoresis method, if necessary, to enhance the penetration into the site where the antigen-presenting cell is present. The peptide of the present invention can be incorporated into various drinks and foods such as sugar tablets, candies, soft drinks, etc., and can be taken orally, or the peptide can be administered through a mucous membrane as desired. Further, the peptide of the present invention can be expressed in vivo by a method such as so-called gene therapy in which RNA encoding the peptide of the present invention is directly administered to an organism and DNA is introduced into a cell.

The method of administering the peptide of the present invention or the composition containing the same to a human body is not particularly limited as long as the peptide of the present invention can be reliably delivered to the site of administration. For example, the composition may be applied dropwise to a mucous membrane in an appropriate amount using a dropper or a syringe, may be taken orally, may be applied to a mucous membrane in the form of a paste or gel, may be introduced into an administration site through a catheter or the like, or may be blown off into a mist form by a nebulizer, a spray or the like, and may be inhaled into a nasal cavity, a trachea or a lung. Administration into a body cavity such as subcutaneous, intradermal, intramuscular, intravascular, intraperitoneal or intrathoracic administration can be carried out by a method of administration such as a syringe, catheter or drip. The dose of the peptide of the present invention may be determined as appropriate in consideration of the antibody-inducing ability, the type of disease, the administration route, the administration method, the animal to be administered, and the like, and is usually 0.00001 to 100mg/kg body weight, preferably 0.0001 to 25mg/kg body weight, and more preferably 0.001 to 10mg/kg body weight. The peptide of the present invention can effectively induce an anti-a β antibody when administered for the first time, and thereafter, can enhance the production of an anti-a β antibody even when additionally immunized with a peptide having only the amino acid sequence of the B cell epitope of the peptide. The peptide of the present invention is described in more detail below based on examples.

< experimental example 1: effect of anti-A.beta.antibody production on B cell epitopes >

Experiments to study the effect of anti-a β antibody production on B cell epitopes were performed as follows.

< preparation of peptide for antibody production >

A peptide having the amino acid sequence at position 298-312 of the Gag protein derived from HIV-1 (SEQ ID NO: 4 in the sequence Listing) was used as a T cell epitope (hereinafter referred to simply as "Gag"). Further, as the B cell epitope, a β (1 to 11) having an amino acid sequence of 1 to 11 th positions from the amino acid terminal of a β (sequence number 1 or 2 in the sequence listing) (sequence number 5 in the sequence listing), a β (1 to 13) having an amino acid sequence of 1 to 13 th positions (sequence number 6 in the sequence listing), and a β (3 to 11) having an amino acid sequence of 3 to 11 th positions (sequence number 8 in the sequence listing) were used. The cell binding motif at the amino terminus of the T cell epitope may be linked to arginine-glycine-aspartic acid (RGD), and to the carboxy terminus thereof, lysine-lysine (KK); peptides represented in Table 1, namely RGD-Gag-KK-A β (3-11) (SEQ ID NO: 32) in the sequence Listing, RGD-Gag-KK-A β (1-11) (SEQ ID NO: 33) in the sequence Listing, and RGD-Gag-KK-A β (1-13) (SEQ ID NO: 34 in the sequence Listing), were synthesized by linking A β (1-11), A β (1-13), or A β (3-11) at the carboxyl terminal thereof as a B cell epitope. The Peptide described in the present specification was synthesized by Fmoc method using a Peptide Synthesizer (model 1350 Multiple Peptide Synthesizer, manufactured by Advanced Chemtech Co., Ltd.), and purified by reverse HPLC using a TSK-GEL column (column diameter: 1cm, length 30cm, manufactured by Kanto Co., Ltd.) to a purity of 95% or more (SIGMAGANGYS division, Peptide purity about 95%).

< measurement of in vivo immunization in mice and antibody titer >

After the above peptide was intranasally administered to BALB/c mice (commercially available from SLC of Japan; female; 5 weeks old; one group; 5 mice), the production amounts (antibody titers) of anti-Gag antibody and anti-A.beta.antibody were measured in the blood of the mice, respectively. Immunization was performed as follows: peptides were administered 4 times per two weeks under the condition of 50. mu.g/mouse/time, blood was collected from each mouse from the last administration to 1 week, and the antibody titers of anti-Gag antibody and anti-Abeta antibody contained in the serum were measured by a common ELISA method using Gag and Abeta (SEQ ID NO: 2 in the sequence listing) as coating antigens. The results are shown in table 1. In the experiment described herein, the antibody titer of each antigen was measured by measuring the amount of the antibody in a standard sample obtained by successively diluting the serum collected from each mouse twice by two times, respectively, by ELISA using an enzyme-linked antibody, measuring the absorbance at 405nm in each well of a microtiter plate used for analysis by a microplate reader (manufactured by Labosystem, inc.), comparing the absorbance with the absorbance in a control well not using the coated antigen, determining the maximum dilution ratio of the serum having a difference of 0.1 or more, and averaging the dilution ratios to represent the antibody titer. In addition, serum from an unimmunized mouse was used as a negative control.

TABLE 1

As can be seen from table 1, the 3 peptides used for immunization had low antigenicity in any case, and the antibody titer results were as follows: the anti-Gag antibody used as a T cell epitope was 11 to 16, and the anti-a β antibody used as a B cell epitope was 14.9 to 32, and the titer of the antibody was about 2 to 3 times lower than that of the non-administered serum (control) in each case, and there was little difference from the background level, so that the above peptide was not suitable for inducing specific antibodies against a β from this experimental system.

< experiment 2: effect of T cell memory on the production of antibodies specific for amyloid beta peptide >

Experiments to investigate the effect of T cell memory on the production of specific antibodies against amyloid β peptide were performed as follows. That is, in the system of experiment 1, the induction of antibody production against T-cell epitopes and B-cell epitopes was hardly detected, in agreement with the background level. Here, when a memory T cell against a T cell epitope is induced in advance and then immunized with the peptide of the present invention, the antibody titer in mouse serum is measured in the same manner as in experiment 1. In addition, when the peptide of the present invention is considered to be used in a human body, since it is not practical to induce memory T cells against antigens (such as Gag) to which humans are rarely sensitized, T cell epitopes derived from tetanus toxoid and diphtheria endotoxin, in which memory T cells exist and immune memory has been established in most humans by vaccination, have been used in this experiment. The T cell epitopes contained in the endotoxins were investigated using a known HLA-DR restriction prediction program (program name "ProPrep") (refer to Singh H. and Raghava G. P. S., "ProPrep: prediction of HLA-DR binding sites", Bioinfomatics, 17 th and 1236 th-1237. 2001), and the multiple antigen recognition epitope regions thereof were investigated, T cell epitopes derived from diphtheria endotoxin were selected as DTL, and T cell epitopes derived from tetanus toxoid were selected as TetT 3L. It was confirmed that any peptide having the amino acid sequence of the T cell epitope could induce differentiation of the T cell against mouse and human multi-restricted epitopes by mixed culture of the peptide and peripheral blood at position 3 of mouse or human with different HLA-DR haplotypes in advance, and was used in this experiment.

< preparation of peptide for inducing antibody production >

With the exception of using DTL or TetT3L as a T cell epitope and a β (1-13) as a B cell epitope, 2 peptides having the same amino acid sequence as the peptide of experiment 1 were synthesized: RGD-DTL-KK-A beta (1-13) (SEQ ID NO: 35 in the sequence Listing), RGD-TetT3L-KK-A beta (1-13) (SEQ ID NO: 36 in the sequence Listing).

< measurement of immunization against mouse and antibody titer >

Before 1 week of administration of the peptides, 50 μ l of 2 mixed vaccines of tetanus and diphtheria (manufactured by Takara Shuzo Chemicals and serotherapy research, containing diphtheria 50Lf and tetanus toxoid in 1ml of about 80 μ g or less) were subcutaneously administered to BALB/c mice (commercially available from SLC, Japan, female, 5 weeks, herd, 5 individuals), to induce memory T cells corresponding to tetanus toxoid and diphtheria endotoxin, and then administered intranasally 4 times at intervals of 2 weeks using 50 μ g/mouse/time of any of the peptides, and blood was collected 1 week after the 4 th administration, and the antibody titers of anti-DTL antibody, anti-TetT 3L antibody, and anti-A β antibody in the blood were measured by the same method as in experiment 1, and the results are shown in Table 2. As a control, no 2 mixed vaccines were administered first, and 3 peptides were immunized in the same dose and by the same method as those in the case of 2 mixed vaccines administered first, and the antibody titer in blood was measured in the same manner, and the results are shown in table 2. In addition, a peptide having an amino acid sequence of DTL, TetT3L, or a β was used as an envelope antigen for antibody titer determination.

TABLE 2

As can be seen from table 2, unlike experiment 1, the administered peptide induced an antibody against a β effectively in any of the mice. When 2 mixed vaccines were administered in advance to induce memory T cells against the T cell epitope of each peptide, the antibody titer was found to increase 64 times when using the T cell epitope derived from diphtheria endotoxin (DTL) and approximately 26 times when using the T cell epitope derived from tetanus toxoid (TetT3L), compared to when no administration was performed. In addition, in the production of antibodies against peptides having amino acid sequences used as T cell epitopes, the occurrence of induction was hardly observed when DTL was used as a T cell epitope. On the other hand, when TetT3L was used, the peptide was also a B cell epitope in mice, and therefore, even though the antibody titer against TetT3L was also increased, the antibody titer due to additional immunization increased by about 2 times that of the 2 mixed vaccines administered in advance, compared to the case where the 2 mixed vaccines were not administered in advance. When 2 or more A.beta. (1-13) s are ligated, the amount of antibody produced can be increased. Approximately the same results were obtained with A.beta. (1-13) replaced with A.beta. (1-15), A.beta. (3-11), and A.beta. (3-15).

The results of this experiment demonstrate that it is possible to prepare a peptide in which immunity has been established in an organism before the peptide of the present invention is administered into the organism, and the production of a β -specific antibodies can be effectively enhanced by using an epitope existing in memory T cells or a peptide containing the same. The experimental results further demonstrate that the induction capability of the production of antibodies against the B cell epitope of the peptide for sensitization can be enhanced by previously administering to an organism for induction memory T cells corresponding to the T cell epitope of the polypeptide for sensitization. Alternatively, DTL is preferred as a T cell epitope when antibody production against the T cell epitope is low. It should be noted that, like the peptide using TetT3L as a T cell epitope, the epitope may be recognized as a B cell epitope due to the haplotype of MHC class II. Although induction of antibody production against epitopes other than the target epitope increases the risk of side effects in the case of a normal peptide vaccine, it has been confirmed that the safety of the peptide of the present invention is extremely high if the T cell epitope is selected within the range of the amino acid sequence of the peptide for vaccination, since the T cell epitope is originally intended to induce production of antibodies against the epitope in a human body, and if the antibody against the T cell epitope is induced, the problem of side effects is not caused.

The present invention will be described in further detail with reference to the following examples, which should not be construed as limiting the technical scope of the present invention in any way. The amounts of the components to be blended in the following examples are shown as the ratio (%) of the whole substance to be blended unless otherwise specified.

Example 1

< composition for enhancing anti-A.beta.antibody production >

Using a T cell epitope (MptL (43-62)) derived from MPT, a secretory protein derived from Mycobacterium tuberculosis, and Abeta (1-13), prepared according to the method of experiment 1, dissolved in distilled water, and sterilized by a usual method to obtain a syrup, the peptide having the amino acid sequence RGD-IQMSDPAYNINISLPSYYPD-KK-DAEFRHDSGYEVH (SEQ ID NO: 37 in the sequence listing) was 100. mu.g/ml, and alpha, alpha-trehalose (reagent grade, commercially available from the institute of forestry and biochemistry) was brought to a concentration of 40%. The resulting solution was poured into a sterile ampoule in an amount of 2ml, and the ampoule was sealed to prepare a syrup containing the peptide. The product has excellent stability and is limited by a wide range of HLA-DR haplotypes, and therefore, it is effective for many people, particularly tuberculosis-positive people, to develop a vaccine that can effectively enhance the production of anti-Abeta antibodies having an effect of preventing and treating various diseases such as neurodegenerative diseases including Alzheimer's disease by nasal or oral administration. When 2 or more A.beta. (1-13) s are ligated, the amount of antibody produced can be increased. Further, substantially the same effect can be obtained even when A.beta. (1-13) is replaced with A.beta. (1-15), A.beta. (3-11), and A.beta. (3-15).

Example 2

< composition for enhancing anti-A.beta.antibody production >

A peptide having an amino acid sequence RGD-AYNFVESIINLFQVVHNSYN-KK-DAEFRHDSGYEVH (SEQ ID NO: 35 in the sequence listing) prepared in accordance with the method of experiment 1 was dissolved in physiological saline containing 1% (w/v) sucrose as a stabilizer using a T cell epitope (DTL) and Ass (1-13) derived from diphtheria endotoxin so as to reach 10. mu.g/ml, 100. mu.g/ml, or 1,000. mu.g/ml, followed by filtration and sterilization. The resulting solutions were put in sterile ampoules in an amount of 1ml, lyophilized and sealed by a conventional method to prepare peptide-containing preparations. Any of these products can be completely dissolved in 1ml of distilled water for use. The product has excellent stability and is restricted by a wide range of HLA-DR haplotypes, and therefore, it is effective for many people, particularly those vaccinated with diphtheria, to develop a vaccine that can effectively enhance the production of anti-A β antibodies having an effect of preventing and treating various diseases such as neurodegenerative diseases including Alzheimer's disease by nasal or oral administration. When 2 or more A.beta. (1-13) s are ligated, the amount of antibody produced can be increased. Further, substantially the same effect can be obtained even when A.beta. (1-13) is replaced with A.beta. (1-15), A.beta. (3-11), and A.beta. (3-15).

Example 3

< safety test of composition for enhancing production of anti-A.beta.antibody >

The peptide prepared in example 1 or example 2 was diluted with physiological saline containing 0.5% sucrose to reach 12.5mg/ml, and then administered to DDY-line male mice at 5 weeks after birth by a conventional method, orally, intraperitoneally, or intramuscularly, to study LD₅₀. LD of any of the above standard₅₀As a result of the fact that the peptide of the present invention is not less than 100mg/kg of mouse body weight in terms of the mass of the peptide, it is considered that the peptide of the present invention is nontoxic and is a safe preparation even when administered to humans.

Example 4

< composition for enhancing anti-A.beta.antibody production >

Using T-cell epitopes derived from tetanus endotoxin (TetT1L) and A.beta.1-13) as T-cell epitopes, a physiological saline solution containing 150. mu.g/ml of a peptide having an amino acid sequence RGD-FLQTMVKLFNRIKNNVAG-KK-DAEFRHDSGYEVH (SEQ ID NO: 38 in the sequence Listing) and 100mg/ml of mannitol was prepared in accordance with the method of experiment 1. They were each poured into a 5ml ampoule to a 1 ml/ampoule and freeze-dried in the conventional manner. The product is restricted by a wide range of HLA-DR haplotypes, and can be administered through mucosa or transdermal membrane of human body, and can be administered to many people, especially to people who have been vaccinated with tetanus, through nasal cavity or oral cavity, to exert the effect of a vaccine capable of effectively enhancing the production of anti-Abeta antibodies having the effect of preventing and treating various diseases such as neuropathy diseases including Alzheimer's disease. When 2 or more A.beta. (1-13) s are ligated, the amount of antibody produced can be increased. Further, substantially the same effect can be obtained even when A.beta. (1-13) is replaced with A.beta. (1-15), A.beta. (3-11), and A.beta. (3-15).

Example 5

< composition for enhancing anti-A.beta.antibody production >

Using T-cell epitopes derived from tetanus endotoxin (TetT3L) and A.beta.1-13) as T-cell epitopes, a physiological saline solution containing 75. mu.g/ml of a peptide having an amino acid sequence RGD-QYIVNEDKFQILYNSIMYGF-KK-DAEFRHDSGYEVH (SEQ ID NO: 36 in the sequence Listing) and 0.5mg/ml of human albumin was prepared in accordance with the method of experiment 1. They were each poured into a 5ml ampoule to a 1 ml/ampoule and freeze-dried in the conventional manner. The product is restricted by a wide range of HLA-DR haplotypes, and therefore, it is effective for many people, particularly for people vaccinated with tetanus, to develop a vaccine that can effectively enhance the production of anti-A β antibodies that have the effect of preventing and treating various diseases such as neuropathic diseases including Alzheimer's disease by mucosal or transdermal administration. When 2 or more A.beta. (1-13) s are ligated, the amount of antibody produced can be increased. Further, substantially the same effect can be obtained even when A.beta. (1-13) is replaced with A.beta. (1-15), A.beta. (3-11), and A.beta. (3-15).

Industrial applicability

As described above, the present invention relates to a peptide that contains an amino acid sequence of a T cell epitope and a B cell epitope having a β, which have been established in the human body for immunological memory, or a peptide containing the amino acid sequence, and that can specifically enhance the production of an antibody specific to a β even in the absence of an immunological adjuvant, and a composition containing the peptide. In addition, since the peptide of the present invention is limited by a wide range of HLA-DR haplotypes, it can be used as a peptide vaccine for inducing the production of anti-A β antibodies having an effect of preventing and treating various diseases such as neurodegenerative diseases including Alzheimer's disease in many people, particularly in people who establish immunological memory by vaccination. Furthermore, the peptide vaccine of the present invention can be used in a form of transmucosal administration such as nasal administration or oral administration without using an immunological adjuvant, and is a simple and safe peptide vaccine as compared with transdermal administration by injection or the like. The invention has obvious effect, makes great contribution to the field and is really a meaningful invention.

Sequence listing

<110>Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo

<120> peptide vaccine for inducing anti-amyloid beta peptide antibody production

<130>WO1132

<160>38

<210>1

<211>40

<212>PRT

<213> human

<223> amyloid beta (1-40)

<400>1

<210>2

<211>42

<212>PRT

<213> human

<223> amyloid beta (1-42)

<400>2

<210>3

<211>20

<212>PRT

<213> Artificial sequence

<223> overlapping multiple restriction site peptide

<400>3

<210>4

<211>15

<212>PRT

<213> human immunodeficiency virus

<223> gag protein (partial sequence)

<400>4

<210>5

<211>11

<212>PRT

<211> human

<213> human

<223> amyloid beta (N-terminal part sequence (1-11))

<400>5

<210>6

<211>13

<212>PRT

<213> human

<223> amyloid beta (N-terminal part sequence (1-13))

<400>6

<210>7

<211>15

<212>PRT

<213> human

<223> amyloid beta (N-terminal part sequence (1-15))

<400>7

<210>8

<211>9

<212>PRT

<213> human

<223> amyloid beta (N-terminal part sequence (3-11))

<400>8

<210>9

<211>13

<212>PRT

<213> human

<223> amyloid beta (N-terminal part sequence (3-15))

<400>9

<210>10

<211>20

<212>PRT

<213> Corynebacterium diphtheriae

<223> diphtheria toxoid (partial sequence)

<400>10

<210>11

<211>19

<212>PRT

<213> Corynebacterium diphtheriae

<223> diphtheria toxoid (partial sequence)

<400>11

<210>12

<211>16

<212>PRT

<213> Clostridium botulinum (Clostridium tenani)

<223> tetanus toxin (partial sequence)

<400>12

<210>13

<211>18

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>13

<210>14

<211>16

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>14

<211>18

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>15

<210>16

<211>18

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>16

<210>17

<211>20

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>17

<210>18

<211>15

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>18

<210>19

<211>16

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>19

<210>20

<211>15

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>20

<210>21

<211>17

<212>PRT

<213> Clostridium botulinum

<223> tetanus toxin (partial sequence)

<400>21

<210>22

<211>17

<212>PRT

<213> Mycobacterium tuberculosis

<223> MPT64 (partial sequence)

<400>22

<210>23

<211>16

<212>PRT

<213> Mycobacterium tuberculosis

<223> MPT64 (partial sequence)

<400>23

<210>24

<211>16

<212>PRT

<213> Mycobacterium tuberculosis

<223> MPT64 (partial sequence)

<400>24

<210>25

<211>16

<212>PRT

<213> Mycobacterium tuberculosis

<223> MPT64 (partial sequence)

<400>25

<210>26

<211>5

<212>PRT

<213> human

<223> binding motif for cell adhesion molecule

<400>26

<210>27

<211>4

<212>PRT

<213> human

<223> binding motif for cell adhesion molecule

<400>27

<210>28

<211>5

<212>PRT

<213> human

<223> binding motif for cell adhesion molecule

<400>28

<210>29

<211>5

<212>PRT

<213> human

<223> binding motif for cell adhesion molecule

<400>29

<210>30

<211>8

<212>PRT

<213> human

<223> binding motif for cell adhesion molecule

<400>30

<210>31

<211>5

<212>PRT

<213> human

<223> binding motif for cell adhesion molecule

<400>31

<210>32

<211>29

<212>PRT

<213> Artificial sequence

<223> peptides for producing anti-amyloid B peptide antibodies

<400>32

<210>33

<211>31

<212>PRT

<213> Artificial sequence

<223> peptides for producing anti-amyloid B peptide antibodies

<400>33

<210>34

<211>33

<212>PRT

<213> Artificial sequence

<223> peptides for producing anti-amyloid B peptide antibodies

<400>34

<210>35

<211>38

<212>PRT

<213> Artificial sequence

<223> peptides for producing anti-amyloid B peptide antibodies

<400>35

<210>36

<211>38

<212>PRT

<213> Artificial sequence

<223> peptides for producing anti-amyloid B peptide antibodies

<400>36

<210>37

<211>38

<212>PRT

<213> Artificial sequence

<223> peptides for producing anti-amyloid B peptide antibodies

<400>37

<210>38

<211>36

<212>PRT

<213> Artificial sequence

<223> peptides for producing anti-amyloid B peptide antibodies

<400>38

Claims (5)

1. A peptide having an amino acid sequence represented by the following general formula, wherein T represents an amino acid sequence containing an amino acid sequence of a T cell epitope for which immunological memory has been established, L represents an amino acid sequence of a linker peptide, B represents an amino acid sequence containing an amino acid sequence of a B cell epitope for which 1 or 2 or more amyloid beta peptides are present, and R is₁、R₂、R₃And R₄At least 1 of which represents an amino acid sequence of a peptide of a cell binding motif of a cell adhesion molecule, of the general formula:

R₁-T-R₂-L-R₃-B-R₄。

2. the peptide according to claim 1, wherein the T cell epitope has an amino acid sequence of 1 or 2 or more peptides selected from tuberculosis, diphtheria, tetanus, and pertussis.

3. A composition which comprises the peptide according to claim 1 or 2 and 1 or more pharmaceutically acceptable additives for formulation and is capable of inducing an antibody against amyloid β peptide by transmucosal administration.

4. The composition as claimed in claim 3, which is used for the prevention and/or treatment of Alzheimer's disease.

5. A DNA encoding the peptide according to claim 1 or 2.