JP2017040544A - Novel biomarker and novel treatment vaccine for type 1 diabetes mellitus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an inspection method of type 1 diabetes mellitus and a vaccine for prevention or treatment of the type 1 diabetes mellitus.SOLUTION: There is provided an inspection method of type 1 diabetes mellitus containing measurement of an autoantibody of (1) or (2), from a sample derived from a subject: (1) is an autoantibody with respect to a peptide formed of an amino acid sequence indicated in a sequence number:2 or a peptide formed of an amino acid sequence corresponding to a sequence number:2 in a nonhuman mammal; and (2) is an autoantibody with respect to a peptide formed of an amino acid sequence indicated in a sequence number:2, or a peptide formed of an amino acid sequence corresponding to a sequence number:2 in a nonhuman mammal, in the amino acid sequence, one or several amino acid residues are substituted, deleted, inserted or added.SELECTED DRAWING: None

Description

  The present invention relates to a method for testing type 1 diabetes comprising measuring an autoantibody against a specific partial amino acid sequence of GFAP (glial fibrillary acidic protein), type 1 diabetes containing the specific partial amino acid sequence of GFAP as an immunogen, and The present invention relates to a vaccine for preventing or treating a disease associated with type 1 diabetes.

  Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which pancreatic islet inflammation and damage cause a shortage of insulin and persistent hyperglycemia, and its incidence is increasing every year. One therapeutic approach for type 1 diabetes is antigen-specific immunotherapy, and (pro) insulin, GAD65, protein tyrosine kinase IA1 (IA-2), HSP60 and the like are listed as target antigens for type 1 diabetes. Currently, therapeutic approaches targeting specific autoantigens (GAD65, IA-2, ZnT8, etc.) presented on pancreatic islets are being conducted in model animal studies and human clinical studies. By shifting T cell differentiation to TH2 bias, we have succeeded in restoring T cells that are self-tolerant to islet cells. In the future, it is anticipated that a combination of immunotherapy targeting different pathways of the immune system will be required to induce immune tolerance in type 1 diabetes.

By the way, recent studies have reported that self-antigens are remarkably expressed not only in islets but also in neuroendocrine tissues such as Schwann cells (pSCs) around the islets. For example, in type 1 diabetes model mice (NOD (non-obese diabetic) mice), inflammation of Schwann cells precedes pancreatic β-cell damage, which is a Schwann cell-specific autoreactive T cell response and Schwann cells. It has been reported that autoantibodies against GFAP expressed in the art are related (Non-patent Document 1). Further, the searched T cell epitopes of GFAP using NOD mice, as peptide antigen presented by K ^d, 79-87 amino acid sequence and 253-261 amino acid sequence of mouse GFAP, antigen presentation by IA ^g7 96-110th amino acid sequence, 116-130th amino acid sequence and 216-230th amino acid sequence of mouse GFAP were found as the peptides to be used, among which the mouse GFAP 79-87th amino acid sequence was used. It has been reported that immunotherapy suppressed the progression of diabetes (Non-patent Document 2). In addition, type 1 diabetes is a T-cell-dependent tissue-specific autoimmune disease, while B cells are also involved in its development, and human blood autoantibody concentration is an important predictive marker for potential autoimmunity. sell. Therefore, a sensitive antibody titer detection system for GFAP is considered to be useful for early diagnosis of type 1 diabetes, and at the same time is considered to be effective for therapeutic intervention, but such a therapeutic approach is still successful. There is no.

Winer, S., et al. Autoimmune islet destruction in spontaneous type 1 diabetes is not beta-cell exclusive.Nature medicine 9, 198-205 (2003). Tsui, H., et al. Targeting of pancreatic glia in type 1 diabetes.Diabetes 57, 918-928 (2008).

  An object of the present invention is to provide a method for testing type 1 diabetes, which comprises detecting a novel autoantibody against a specific partial amino acid sequence of GFAP. Another object of the present invention is to provide a vaccine for preventing or treating type 1 diabetes or a disease associated with type 1 diabetes, which comprises a specific partial amino acid sequence of GFAP.

The inventors of the present invention observed the anti-GFAP antibody of the NOD mouse that is a type 1 diabetes model mouse over time, and the anti-GFAP antibody increased from an earlier stage than the anti-GAD65 antibody that is an existing type 1 diabetes marker, It was found that anti-GFAP antibody titer and C peptide level showed an inverse correlation. In addition, NOD mice positive for anti-GFAP antibody had a higher incidence of type 1 diabetes than NOD mice positive for anti-GAD65 antibody. Furthermore, the amino acid sequence of mouse GFAP recognized by the anti-GFAP antibody of NOD mouse was identified, an amino acid sequence different from the amino acid sequence was selected, and a vaccine conjugated with KLH was administered to NOD mouse. It was found for the first time that T cell differentiation to the overweight was induced and the onset of type 1 diabetes was suppressed.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] A method for examining type 1 diabetes, comprising measuring the following autoantibody (1) or (2) from a sample derived from a subject:
(1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or an autoantibody against a peptide consisting of the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal, and (2) an amino acid sequence shown in SEQ ID NO: 2. Or an autoantibody against a peptide consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal;
[2] The test method according to [1], wherein the sample is plasma or serum;
[3] The method according to [1] or [2], wherein autoantibodies are measured by an immunological method;
[4] A test kit for type 1 diabetes comprising the following peptide (1) or (2):
(1) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a peptide consisting of the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal, and (2) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2. Or a peptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal;
[5] Type 1 diabetes or a vaccine for prevention or treatment of a disease associated with type 1 diabetes, comprising any of the following substances (1) to (3):
(1) A peptide comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal;
(2) An amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal And (3) an expression vector capable of expressing the peptide of (1) or (2) above;
[6] The vaccine according to [5], comprising a carrier protein;
[7] The vaccine according to [6], wherein the carrier protein is mussel hemocyanin;
[8] The vaccine according to any one of [5] to [7], comprising an adjuvant;
I will provide a.

  By detecting an autoantibody that recognizes a specific amino acid sequence of GFAP from a sample of a subject, it can be accurately determined whether the subject develops type 1 diabetes or will develop in the future. In addition, by using a specific amino acid sequence of GFAP as a vaccine, it suppresses T cell reactivity, and induces T cell differentiation from Th1 to Th2 to induce type 1 diabetes and type 1 diabetes related diseases. Can prevent or treat the onset.

A is a graph showing the incidence of type 1 diabetes in NOD mice (n = 30). B: It is a figure which shows each IgG subclass of the anti- GFAP antibody couple | bonded with full length GFAP protein. **: P <0.01 A: A graph showing the increase ratio of anti-GFAP antibody titer in 9, 17, 21, and 25-week-old NOD mice (n = 30) based on the anti-GFAP antibody titer of 9-week-old NOD mice. B: A graph showing the increase rate of anti-GAD65 antibody titer in 9, 17, 21, and 25-week-old NOD mice (n = 30) based on the anti-GAD65 antibody titer of 9-week-old NOD mice. A: It is a graph which shows the correlation between the increase rate of the anti- GFAP antibody titer of 21 and 25-week-old NOD mouse | mouth, and a glucose level at any time. Glucose levels were measured continuously for 2 days at 27 weeks of age. B: is a graph showing the correlation between the fold increase in anti-GAD65 antibody titer of 21- and 25-week-old NOD mice and the occasional glucose level. Glucose levels were measured continuously for 2 days at 27 weeks of age. A: It is a graph which shows the correlation between the increase rate of the anti- GFAP antibody titer of all the NOD mice of 25 weeks old, and C peptide secretion. B: It is a graph which shows the correlation between the increase magnification of the anti- GAD65 antibody titer of all the NOD mice of 25 weeks old, and C peptide secretion. C is a graph showing the correlation between the increase in anti-GFAP antibody titer and C peptide secretion in 25-week-old NOD mice (diabetic group). D: A graph showing the correlation between the increase in anti-GAD65 antibody titer and C peptide secretion in 25-week-old NOD mice (diabetic group). E: It is a graph which shows the correlation between the increase rate of the anti- GFAP antibody titer of a 25-week-old NOD mouse (non-diabetic group), and C peptide secretion. F: A graph showing the correlation between the increase in anti-GAD65 antibody titer and C peptide secretion in 25-week-old NOD mice (non-diabetic group). A: A graph showing the proportion of mice that were positive for anti-GFAP antibody at each time of 17, 21, and 25 weeks of age developing diabetes at 30 weeks of age. B: It is a graph which shows the ratio which the mouse | mouth which was positive for the anti- GAD65 antibody at each time of 17, 21, and 25 weeks old has diabetes at the time of 30 weeks old. C: A graph showing the percentage of mice that were positive for anti-GFAP antibody and anti-GAD65 antibody at each time of 17, 21, and 25 weeks of age developing diabetes at the time of 30 weeks of age. D: It is a graph which shows the ratio which the mouse | mouth which was negative of the anti- GAD65 antibody and the anti- GAD65 antibody at each time of 17, 21, and 25-week-old has developed diabetes at the 30-week-old time. A: It is a figure which shows the coupling | bonding of each partial peptide C1, C2, C3, C4 of an anti- GFAP antibody and GFAP. ***: P <0.005, B: Each IgG subclass of anti-GFAP antibody binding to C3 peptide. *: P <0.05 A is a graph showing antibody titers of NOD mice immunized with GFAP vaccine, KLH vaccine, or physiological saline. B: is a graph showing the onset of type 1 diabetes in NOD mice immunized with GFAP vaccine, KLH vaccine or saline. A: Graph showing C-peptide levels in NOD mice immunized with GFAP vaccine, KLH vaccine or saline. B: is a graph showing the degree of infiltration of T cells into the islets of NOD mice immunized with GFAP vaccine, KLH vaccine or physiological saline. C is a graph showing the binding of antibodies derived from NOD mice immunized with GFAP vaccine, KLH vaccine, or physiological saline to C1 peptide, C2 peptide, C3 peptide and C4 peptide. *: P <0.05, **: P <0.01, ***: P <0.005; +: P <0.05, ++: P <0.01; #: P <0.05, D: Anti-GFAP antibody binding to C4 peptide It is a figure which shows each IgG subclass. *: P <0.05, **: P <0.01 A: Stimulating spleen cells of NOD mice with type 1 diabetes with C3 peptide, recombinant GFAP (full length), recombinant GAD65 (full length) or PHA, showing IL-4 or IFN-γ-producing cells is there. B: Graph showing the number of cells that produce IL-4 or IFN-γ by stimulating spleen cells of NOD mice with type 1 diabetes with C3 peptide, recombinant GFAP (full length), recombinant GAD65 (full length) or PHA It is. C: Shows cells that produce IL-4 or IFN-γ by stimulating spleen cells of NOD mice that do not develop type 1 diabetes with C3 peptide, recombinant GFAP (full length), recombinant GAD65 (full length) or PHA FIG. D: Number of cells producing IL-4 or IFN-γ by stimulating spleen cells of NOD mice not developing type 1 diabetes with C3 peptide, recombinant GFAP (full length), recombinant GAD65 (full length) or PHA It is a graph to show. A: Tissue staining image of pancreatic islet tissue of NOD mouse immunized with KLH vaccine. B: Spleen cells of NOD mice immunized with KLH vaccine are stimulated with recombinant GFAP (full length), recombinant GAD65 (full length) or PHA to show cells that produce IL-4 or IFN-γ. FIG. 6 is a graph showing the number of cells that produce IL-4 or IFN-γ by stimulating spleen cells of NOD mice immunized with C: KLH vaccine with recombinant GFAP (full length), recombinant GAD65 (full length) or PHA. *: P <0.05, **: P <0.01

1. Method for Examining Type 1 Diabetes As described in Examples below, it was found that autoantibodies having a specific amino acid sequence of mouse GFAP as an epitope serve as a marker for the onset of type 1 diabetes in NOD mice. Therefore, the present invention includes a method for testing type 1 diabetes (hereinafter referred to as the test method of the present invention), which comprises measuring the following autoantibody (1) or (2) from a sample derived from a subject:
(1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or an autoantibody against a peptide consisting of the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal, and (2) an amino acid sequence shown in SEQ ID NO: 2. Or an autoantibody against a peptide consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal Is.

  The subject of the test method of the present invention may be any mammal, but is a mammal that has developed type 1 diabetes or a mammal that is likely to develop type 1 diabetes. Mammals include, for example, rodents such as mice, rats, hamsters, guinea pigs, and laboratory animals such as rabbits, pets such as dogs and cats, livestock such as cows, pigs, goats, horses and sheep, humans, monkeys, Examples include primates such as orangutans and chimpanzees, and humans are particularly preferable. The subject may or may not be receiving treatment for type 1 diabetes.

  The subject-derived sample is not particularly limited, but is preferably one that is less invasive to the subject, and examples thereof include those that can be easily collected from a living body such as blood, plasma, serum, urine, and saliva. When serum or plasma is used, it can be prepared by collecting blood from a subject according to a conventional method and separating the liquid component. In addition, a sample derived from a subject can be subjected to separation and removal of a low molecular weight peptide fraction or the like in advance before carrying out the test method of the present invention.

The autoantibody measured by the test method of the present invention (hereinafter, the autoantibody of the present invention)
(1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or an autoantibody against a peptide consisting of the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal, or (2) an amino acid sequence shown in SEQ ID NO: 2. Or an autoantibody against a peptide consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal.

  The peptide to which the autoantibody of (1) above measured by the test method of the present invention binds is a partial sequence of the amino acid sequence of GFAP (glial fibrillary acidic protein). GFAP is a known gene, and its nucleotide sequence and amino acid sequence are also known. Specifically, as the peptide to which the autoantibody of (1) above binds, in the case of human, the amino acid represented by SEQ ID NO: 2 corresponding to amino acid numbers 279 to 287 of the human GFAP amino acid sequence (SEQ ID NO: 11) Mention may be made of peptides consisting of sequences. The partial sequence is encoded by a nucleotide sequence represented by SEQ ID NO: 1, for example. Furthermore, preferred examples of the peptide to which the antibody of (1) binds include a peptide consisting of an amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal. As an amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal, appropriate primers and probes using the information of the amino acid sequence disclosed in SEQ ID NO: 2 in the present specification and a known sequence database And can be easily obtained using ordinary genetic engineering techniques such as RT-PCR and plaque hybridization. For example, the partial sequence of mouse GFAP amino acid sequence (SEQ ID NO: 12) corresponding to SEQ ID NO: 2 which is a partial sequence of human GFAP can be exemplified as the amino acid sequence represented by SEQ ID NO: 8, The sequence is encoded by the nucleotide sequence represented by SEQ ID NO: 7, for example. Further, the non-human mammal here is a mammal excluding humans from the above-mentioned mammals.

  The test method of the present invention can also be used for a subject having a GFAP having a naturally occurring GFAP mutant or polymorphism (substitution, deletion, insertion, etc. of 1 to several amino acids). Therefore, the peptide to which the autoantibody of the above (2) measured by the test method of the present invention binds is one or several (preferably 1 to several (2-5)) in the partial sequence of the GFAP amino acid sequence. Peptides having an amino acid sequence in which amino acids are deleted, substituted, inserted or added are included. As such a peptide, in the case of human, one or several (preferably 1 to several (2 to 5)) amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 2. A peptide consisting of the amino acid sequence thus prepared is also included. Examples of the amino acid sequence include (1) an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence shown in SEQ ID NO: 2 have been deleted, (2 ) An amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are added to the amino acid sequence shown in SEQ ID NO: 2, and (3) an amino acid sequence shown in SEQ ID NO: 2. An amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids are inserted; (4) one or several (preferably 1 to several) in the amino acid sequence shown in SEQ ID NO: 2 (2) amino acid sequences in which amino acids are substituted with other amino acids, or (5) amino acid sequences in which the mutations in (1) to (4) above are combined (in this case, the sum of the mutated amino acids is 1 or several (preferably 1 to several (2-5)), among them (1) an amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are deleted from the amino acid sequence shown in SEQ ID NO: 2, and (2) SEQ ID NO: 2 An amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are added to the amino acid sequence shown in FIG. The peptide to which the autoantibody of (2) binds is one or several (preferably 1 to several (2-5)) amino acids in the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal. Peptides consisting of amino acid sequences in which is deleted, substituted, inserted or added can also be preferably mentioned. As such a peptide, in the case of a mouse, one or several (preferably 1 to several (2 to 5)) amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4. A peptide consisting of the amino acid sequence thus prepared is also included. Examples of the amino acid sequence include (1) an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 8 have been deleted, (2 ) An amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are added to the amino acid sequence shown in SEQ ID NO: 8, and (3) an amino acid sequence shown in SEQ ID NO: 8. An amino acid sequence into which one or several (preferably 1 to several (2 to 5)) amino acids are inserted; (4) one or several (preferably 1 to several) in the amino acid sequence shown in SEQ ID NO: 8; (2) amino acid sequences in which amino acids are substituted with other amino acids, or (5) amino acid sequences in which the mutations in (1) to (4) above are combined (in this case, the sum of the mutated amino acids is 1 or several (preferably 1 to several (2 to 5))) (1) an amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids in the amino acid sequence shown in SEQ ID NO: 8 have been deleted; and (2) SEQ ID NO: 8 Preferred is an amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are added to the amino acid sequence shown.

  Examples of “amino acid residue substitution” include conservative amino acid substitution. Conservative amino acid substitution refers to substitution of a specific amino acid with an amino acid having a side chain having the same properties as the side chain of the amino acid. Specifically, in a conservative amino acid substitution, a particular amino acid is replaced with another amino acid belonging to the same group as the amino acid. Groups of amino acids having side chains of similar nature are known in the art. For example, such amino acid groups include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having neutral side chains. (For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan). In addition, the amino acid having a neutral side chain further includes an amino acid having a polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), and an amino acid having a nonpolar side chain (for example, alanine, Valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan). In addition, as other groups, for example, amino acids having aromatic side chains (for example, phenylalanine, tryptophan, tyrosine), amino acids having side chains including hydroxyl groups (alcoholic hydroxyl groups, phenolic hydroxyl groups) (for example, serine, threonine, Tyrosine).

  As the “deletion of amino acid residue”, for example, any amino acid residue can be selected and deleted from the amino acid sequence represented by SEQ ID NO: 2, and among them, the N-terminal side or C It is preferable to delete one or several (preferably 1 to several (2 to 5)) amino acid residues on the terminal side. For example, an amino acid in which one or several (preferably 1 to several (2 to 5)) amino acids of the amino acid residue on the N-terminal side and / or C-terminal side of the amino acid sequence shown in SEQ ID NO: 2 have been deleted Examples include sequences.

  As the “insertion of amino acid residue”, for example, an amino acid residue can be inserted into the amino acid sequence represented by SEQ ID NO: 2, at the N-terminal side or C-terminal side.

  As the “addition of amino acid residue”, for example, an amino acid residue can be added to the inside, N-terminal side or C-terminal side of the amino acid sequence represented by SEQ ID NO: 2, and among them, the N-terminal side or It is preferable to add one or several (preferably 1 to several (2 to 5)) amino acid residues on the C-terminal side. For example, an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids are added to the N-terminal side and / or C-terminal side amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 Etc. The amino acid sequence to be added may be any amino acid sequence, but is preferably the corresponding amino acid in the GFAP amino acid sequence (for example, SEQ ID NO: 11 for humans). For example, when 5 amino acids are added to the N-terminal side of SEQ ID NO: 2 corresponding to amino acid numbers 279 to 287 of the human GFAP amino acid sequence (SEQ ID NO: 11), the added amino acid sequence is the amino acid of SEQ ID NO: 11 Preferably, the amino acid sequence corresponds to Nos. 274 to 278, and when 5 amino acids are added to the C-terminal side of SEQ ID NO: 2, the added amino acid sequence corresponds to amino acid numbers 288 to 292 of SEQ ID NO: 11. The amino acid sequence is preferably

The peptide to which the autoantibody of the present invention binds may have a carboxyl terminus, carboxylate, amide or ester at the C-terminus. In addition, when the peptide has a carboxyl group (or carboxylate) other than the C-terminal, the carboxyl group may be amidated or esterified. Further, the peptide has a peptide in which the amino group of the N-terminal amino acid residue is substituted with, for example, a formyl group or an acetyl group, a peptide in which the N-terminal glutamine residue is pyroglutamine oxidized, or the amino acid side in the molecule. Even if a substituent on the chain (for example, --OH, --SH, amino group, imidazole group, indole group, guanidino group, etc.) is substituted with another substituent (for example, formyl group, acetyl group, etc.) Good.
The peptide may be a salt with an acid or a base, and an acid addition salt is particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

The peptide can be chemically synthesized based on known amino acid sequence information.
The peptide containing a part of the amino acid sequence of the peptide is preferably chemically synthesized based on the above-described amino acid sequence information of GFAP. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the peptide of interest can be produced by condensing a partial peptide or amino acid that can constitute the peptide and the remaining part, and removing the protecting group when the product has a protecting group. Examples of known condensation methods and protecting group removal include the methods described in 1) or 2) below.
1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
Further, after the reaction, the peptide can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it is converted into a free form or other salt by a known method. can do.

  The autoantibody in the test method of the present invention can be measured using a peptide recognized by the autoantibody, an antibody recognizing the peptide recognized by the autoantibody, a secondary antibody against the autoantibody, and the like.

  As the peptides recognized by the autoantibodies of the present invention, the same peptides as those described above can be used.

  The antibody recognizing the peptide recognized by the autoantibody of the present invention is not particularly limited as long as it specifically recognizes the peptide recognized by the autoantibody of the present invention. In addition to the complete antibody molecule, for example, Fab, Fab ', F (ab') 2 fragments, scFv, scFv-Fc, genetically engineered conjugate molecules such as minibodies and diabodies, or protein stabilization such as polyethylene glycol (PEG) They may be derivatives thereof modified with molecules.

When the above antibody is a monoclonal antibody, it can be prepared, for example, by the following method.
The above peptide to which the autoantibody of the present invention binds is prepared. The peptide can be directly immunized if it has immunogenicity, but when using a low molecular weight antigen, these antigenic peptides are usually hapten molecules with low immunogenicity. The immunization can be carried out as a complex bound or adsorbed to an appropriate carrier (carrier). A natural or synthetic polymer can be used as the carrier. Examples of natural polymers include serum albumin of mammals such as cows, rabbits and humans, and thyroglobulin of mammals such as cows and rabbits, such as chicken ovalbumin, such as mammals such as cows, rabbits, humans and sheep. Hemoglobin, squash hemocyanin (KLH), etc. are used. Examples of the synthetic polymer include various latexes such as polymers or copolymers such as polyamino acids, polystyrenes, polyacryls, polyvinyls, and polypropylenes. As long as the antibody against the antigen bound or adsorbed to the carrier can be produced efficiently, any carrier can be bound or adsorbed at any ratio. The above-mentioned natural or synthetic polymer carrier, which is commonly used in the production of the above, can be used which is bound or adsorbed at a weight ratio of 0.1 to 100 with respect to hapten 1.

  Various condensing agents can be used for coupling the hapten and the carrier protein. For example, diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldehyde that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and thiol groups A dimaleimide compound such as N, N′-o-phenylene dimaleimide, a maleimide active ester compound that crosslinks an amino group and a thiol group, a carbodiimide compound that crosslinks an amino group and a carboxyl group, and the like are advantageously used. In addition, when amino groups are cross-linked, an active ester reagent having a dithiopyridyl group (for example, SPDP) is reacted with one amino group and then reduced to introduce a thiol group, and the other amino group It is also possible to react both after introducing a maleimide group with a maleimide active ester reagent. It is also possible to add a cysteine residue to the N-terminus or C-terminus of the hapten (peptide), introduce a maleimide group into the amino group of the carrier protein with a maleimide active ester reagent, and then react both.

The antigenic peptide is administered to a warm-blooded animal by itself, for example by intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, or the like alone or together with a carrier or diluent at a site where antibody production is possible. The Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually performed once every 1 to 6 weeks, about 2 to 10 times in total. Examples of warm-blooded animals include rabbits, goats, cows, chickens, mice, rats, hamsters, sheep, pigs, horses, camels, cats, dogs, monkeys, chimpanzees, etc. Rats, rabbits and the like are preferably used.
In the production of monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with a confirmed serum antibody titer from a mouse, and remove the spleen or lymph nodes 2 to 5 days after the final immunization, A monoclonal antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained therein with myeloma cells. The antibody titer in the antiserum is measured, for example, by reacting the immobilized antigen peptide with the antiserum, and then immunizing animal species in which the antigen peptide-specific antibody bound to the solid phase is labeled with a radioactive substance or enzyme. It can carry out by detecting with the antibody with respect to the antibody of. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc., and P3U1 is preferably used. The preferred ratio of the number of antibody-producing cells (spleen cells) to be used and the number of myeloma cells is about 1: 1-20: 1, and PEG (preferably PEG1000-PEG6000) is added at a concentration of about 10-80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.

Selection of fused cells (hybridomas) can be carried out according to a method known per se or a method analogous thereto, but can usually be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium may be used as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free for hybridoma culture Medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually 20-40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas.
Various methods can be used to screen for monoclonal antibody-producing hybridomas.For example, the hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which an antigen peptide is adsorbed directly or together with a carrier, and then a radioactive substance or A method for detecting a monoclonal antibody bound to a solid phase by adding an anti-immunoglobulin antibody labeled with an enzyme or the like (if the antibody-producing cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A; Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which an anti-immunoglobulin antibody or protein A is adsorbed, an antigen peptide labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected. It is done.

  Separation and purification of monoclonal antibodies can be carried out in the same way as separation and purification of ordinary polyclonal antibodies (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchanger (eg, DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method to obtain antibody by dissociating the binding using active adsorbent such as protein A or protein G) Can be done according to

When the above antibody is a polyclonal antibody, it can be produced according to a method known per se or a method analogous thereto. For example, the complex of the peptide and carrier protein is immunized to a warm-blooded animal in the same manner as the above-described method for producing a monoclonal antibody, and an antibody-containing product against the antigen is collected from the immunized animal, and the antibody is separated and purified. Can be manufactured.
Regarding the complex of hapten and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio of carrier and hapten should be such that an antibody can be efficiently produced against a hapten that has been cross-linked to a carrier and immunized. Any ratio may be cross-linked at any ratio, for example, bovine serum albumin, bovine thyroglobulin, KLH, etc., in a weight ratio of about 0.1 to 20, preferably about 1 to 5, with respect to hapten 1. A method of combining in proportion is used.
In addition, various condensing agents can be used for coupling of the hapten and the carrier protein, but an active ester reagent containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, dithiobilidyl group, or the like is used.
The condensation product is administered to warm-blooded animals at the site where antibody production is possible, or with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration can be performed about once every 2 to 6 weeks, usually about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, breast milk, eggs, etc. of warm-blooded animals immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

  Examples of the secondary antibody against the autoantibody of the present invention include secondary antibodies and aptamers against the antibody. The aptamer against the autoantibody of the present invention can be obtained by a SELEX technique known per se. On the other hand, the secondary antibody against the autoantibody of the present invention is the above-mentioned “peptide recognized by the autoantibody of the present invention” using the autoantibody or a fragment thereof (eg, F (ab ′) 2, Fab) as an immunogen. It can be obtained according to the method described. IgG1, IgG2b, IgG2c, and the like are listed as subclasses of antibodies used as immunogens, and IgG2b is preferred. The autoantibodies of the present invention to be used for the SELEX method and animal immunization can be isolated from serum, plasma, etc. derived from patients with type 1 diabetes using the affinity for the aforementioned peptides. A desired fragment can be obtained by digesting the resulting autoantibody with pepsin or papain.

  The test method of the present invention is not particularly limited, and the amount of antigen, antibody or antigen-antibody complex corresponding to the amount of autoantibodies in the test sample is detected by chemical or physical means, and this is detected. Any measurement method may be used as long as it is an immunological measurement method calculated from a standard curve prepared using a standard solution containing a known amount of antibody. For example, nephrometry, competition method, immunometric method, sandwich method, western blotting, SPR, turbidimetric method and the like are preferably used.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably stable and has a large specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.

  For insolubilization of an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

  In the sandwich method, a sample derived from a subject is reacted with an insolubilized peptide recognized by an autoantibody (primary reaction), and a labeled secondary antibody or labeled antigen against the autoantibody is further reacted (secondary reaction). By measuring the amount (activity) of the labeling agent on the insolubilized carrier, the amount of the autoantibody of the present invention in the sample can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.

In the competitive method, an autoantibody in a sample derived from a subject and a labeled antibody that recognizes the same antigen are competitively reacted with an antigen peptide, and then the unreacted labeled antibody (F) is bound to the antigen. Separate the labeled antibody (B) (B / F separation), measure the amount of either B or F, and quantify the amount of autoantibodies in the sample.
In the immunometric method, the solid antibody and the liquid phase are separated after subjecting the autoantibody of the sample derived from the subject and the immobilized antibody recognizing the same antigen to competitive reaction with a certain amount of labeled antigen, or Then, the autoantibody in the sample is reacted with an excess amount of labeled antigen, and then the immobilized antibody is added to bind the unreacted labeled antigen to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of labeling in either phase is measured to quantify the amount of autoantibodies in the sample.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of autoantibodies in the sample derived from the subject is small and only a small amount of precipitate is obtained.

  Alternatively, using a surface plasmon resonance (SPR) method, a peptide recognized by an autoantibody is immobilized on the surface of a commercially available sensor chip (eg, manufactured by Biacore) according to a conventional method, and a sample derived from a subject is brought into contact therewith. Thereafter, the sensor chip is irradiated with light of a specific wavelength from a specific angle, and the presence or absence of the binding of the autoantibody to the immobilized peptide can be determined using the change in the resonance angle as an index. Alternatively, the peptide recognized by the autoantibody is immobilized on the surface of a probe that can be adapted to a mass spectrometer as described above, the sample is brought into contact with the peptide on the probe, and the sample components captured by the peptide are collected. The autoantibody of the present invention can also be measured by a method of detecting a peak corresponding to the molecular weight of the autoantibody recognizing the peptide by mass spectrometry.

  When the level of the autoantibody of the present invention in the sample derived from the subject measured by any of the above methods is significantly increased compared to the level of the marker in the control sample derived from a healthy subject, It can be diagnosed that there is a high possibility of suffering from type 1 diabetes or a high possibility of suffering in the future.

  When the test method of the present invention is applied to a patient who has already developed type 1 diabetes, samples are collected in time series from patients with type 1 diabetes, and the time course of the expression of the autoantibody of the present invention in each sample It is preferable to carry out by examining. The sampling interval is not particularly limited, but it is desirable to sample as frequently as possible within a range that does not impair the patient's QOL. When the level of these markers decreases with time, it can be determined that there is a high possibility that the pathology of type 1 diabetes in the patient is improved.

  Furthermore, in the method for testing type 1 diabetes by the above time-series sampling, when a treatment measure for the disease is taken for the subject patient between the previous sampling and the current sampling, the treatment by the measure is performed. It can be used to evaluate the effect. That is, for a sample sampled before and after treatment, when it is determined that the condition after treatment is improved compared to the condition before treatment, it can be evaluated that the treatment is effective. On the other hand, when it is determined that the condition after treatment is not improved or further deteriorated as compared with the condition before treatment, it can be evaluated that the treatment has no effect.

2. Vaccine for preventing or treating type 2 diabetes As shown in the examples described later, by using a partial peptide of GFAP having a different amino acid sequence from the epitope of the autoantibody of the present invention as a vaccine, blood C peptide level in NOD mice And the onset of T-cell infiltration into the islets were able to delay the onset of type 1 diabetes. Therefore, the present invention also provides a vaccine for preventing or treating type 1 diabetes and a disease associated with type 1 diabetes, which comprises any of the following substances (1) to (3):
(1) A peptide comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal;
(2) An amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal And (3) an expression vector capable of expressing the peptide of (1) or (2) above.

  Diseases to be prevented or treated by the vaccine of the present invention include diseases associated with type 1 diabetes in addition to type 1 diabetes. Examples of diseases associated with type 1 diabetes include diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, hyperinsulinemia, obesity and the like.

  The subject of administration of the vaccine of the present invention may be the same as the subject of the test method of the present invention. In addition, when the vaccine of the present invention is administered, the substance contained in the vaccine is a substance derived from the administration subject (that is, when administered to a human, the vaccine is a human-derived substance, and when administered to a mouse, The vaccine is preferably a mouse-derived substance).

Substances contained in the vaccine of the present invention are:
(1) A peptide comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal;
(2) An amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal And (3) a substance selected from the group consisting of expression vectors capable of expressing the peptide of (1) or (2) above.

  The peptide of the above (1) contained in the vaccine of the present invention (hereinafter, the peptide of the present invention together with the peptide of the above (2)) is a partial sequence of the amino acid sequence of GFAP. Examples of the peptide of (1) above include, in the case of humans, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 corresponding to amino acid numbers 417 to 430 of the human GFAP amino acid sequence (SEQ ID NO: 11). . The partial sequence is encoded by a nucleotide sequence represented by SEQ ID NO: 3, for example. Furthermore, as a peptide of said (1), the peptide which consists of an amino acid sequence corresponding to sequence number: 4 in a non-human mammal can also be mentioned preferably. As an amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal, an appropriate primer or probe is used by utilizing information on the amino acid sequence disclosed in SEQ ID NO: 4 in the present specification or a known sequence database. And can be easily obtained using ordinary genetic engineering techniques such as RT-PCR and plaque hybridization. For example, the partial sequence of the mouse GFAP amino acid sequence (SEQ ID NO: 12) corresponding to SEQ ID NO: 4 which is a partial sequence of human GFAP can be exemplified as the amino acid sequence represented by SEQ ID NO: 10; The sequence is encoded by the nucleotide sequence represented by SEQ ID NO: 9, for example. Further, the non-human mammal here is a mammal excluding humans from the above-mentioned mammals.

  The vaccine of the present invention can also be used for a subject having a GFAP having a naturally occurring GFAP mutant or polymorphism (substitution, deletion, insertion, etc. of 1 to several amino acids). Therefore, as the peptide of (2) contained in the vaccine of the present invention, one or several (preferably 1 to several (2-5)) amino acids are deleted or substituted in the partial sequence of the GFAP amino acid sequence. Peptides consisting of an inserted or added amino acid sequence can be mentioned. As such a peptide, in the case of humans, one or several (preferably 1 to several (2-5)) amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 4. A peptide consisting of the amino acid sequence thus prepared is also included. Examples of the amino acid sequence include (1) an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 4 have been deleted, (2 ) An amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are added to the amino acid sequence shown in SEQ ID NO: 4, and (3) an amino acid sequence shown in SEQ ID NO: 4. An amino acid sequence into which one or several (preferably 1 to several (2 to 5)) amino acids are inserted; (4) one or several (preferably 1 to several) in the amino acid sequence shown in SEQ ID NO: 4; (2) amino acid sequences in which amino acids are substituted with other amino acids, or (5) amino acid sequences in which the mutations in (1) to (4) above are combined (in this case, the sum of the mutated amino acids is 1 or several (preferably 1 to several (2 to 5))) (1) an amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids in the amino acid sequence shown in SEQ ID NO: 4 have been deleted; and (2) SEQ ID NO: 4 Preferred is an amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are added to the amino acid sequence shown. In addition, as the peptide (2), one or several (preferably 1 to several (2 to 5)) amino acids are deleted or substituted in the amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal. A peptide comprising an inserted or added amino acid sequence can also be preferably exemplified. As such a peptide, in the case of a mouse, one or several (preferably 1 to several (2-5)) amino acids are deleted, substituted, inserted or added in the amino acid sequence represented by SEQ ID NO: 10. A peptide consisting of the amino acid sequence thus prepared is also included. Examples of the amino acid sequence include (1) an amino acid sequence in which one or several (preferably 1 to several (2 to 5)) amino acids in the amino acid sequence represented by SEQ ID NO: 10 have been deleted, (2 ) An amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are added to the amino acid sequence shown in SEQ ID NO: 10, and (3) an amino acid sequence shown in SEQ ID NO: 10. An amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are inserted; (4) one or several (preferably 1 to several) in the amino acid sequence shown in SEQ ID NO: 10; (2) amino acid sequences in which amino acids are substituted with other amino acids, or (5) amino acid sequences in which the mutations in (1) to (4) above are combined (in this case, the sum of the mutated amino acids is 1 or several (preferably 1 to several (2-5))), Among them, (1) an amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids in the amino acid sequence shown in SEQ ID NO: 10 have been deleted, and (2) SEQ ID NO: Preferred is an amino acid sequence in which one or several (preferably 1 to several (2-5)) amino acids are added to the amino acid sequence shown in FIG.

  As the “deletion, substitution, insertion or addition of amino acid residues”, the description relating to the deletion, substitution, insertion or addition of the amino acid sequence described in the test method of the present invention may be used.

  The peptides of the present invention may contain additional amino acids. Such amino acid additions are permissible as long as the peptide induces a specific immune response against GFAP. The amino acid sequence to be added is not particularly limited, and examples thereof include a tag for facilitating detection and purification of the peptide. Tags include Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (eg GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. It can be illustrated. The position to which the amino acid sequence is added is the N-terminal and / or C-terminal of the peptide of the present invention.

  The amino acids used in the peptide of the present invention include L-form, D-form and DL-form, but usually L-form is preferred. These peptides can be synthesized by a normal peptide synthesis method and used in the present invention, but the production method, synthesis method, procurement method, and the like are not particularly limited in the present invention.

In the expression vector of (3) above, the nucleotide (DNA or RNA, preferably DNA) encoding the peptide of (1) or (2) above exhibits promoter activity in the mammalian cell to be administered. Is operably linked downstream of a possible promoter. That is, the expression vector (3) can express the peptide (1) or (2) as a transcription product under the control of a promoter. By administering the expression vector of (3) to a mammal, the peptide of (1) or (2) is produced in the body of the mammal, and the mammal is specific for the peptide of (1) or (2). An immune response is induced.
The promoter to be used is not particularly limited as long as it can function in mammalian cells to be administered. As the promoter, a pol I promoter, pol II promoter, pol III promoter, or the like can be used. Specifically, SV40-derived early promoter, viral promoter such as cytomegalovirus LTR, mammalian constituent protein gene promoter such as β-actin gene promoter, and the like are used.

  The expression vector (3) preferably contains a transcription termination signal, that is, a terminator region, downstream of the nucleotide encoding the peptide (1) or (2). Furthermore, a selection marker gene for selecting transformed cells (a gene that imparts resistance to drugs such as tetracycline, ampicillin, and kanamycin, a gene that complements an auxotrophic mutation, and the like) can be further contained.

  The type of vector used for the expression vector in the present invention is not particularly limited, and examples of vectors suitable for administration to mammals such as humans include viral vectors and plasmid vectors. Examples of viral vectors include retroviruses, adenoviruses, adeno-associated viruses, and the like. In view of ease of production and handling and economic efficiency, a plasmid vector is preferably used.

  The vaccine of the present invention can be provided as a pharmaceutical composition containing any carrier, for example, a pharmaceutically acceptable carrier, in addition to the peptide of (1) or (2) or the expression vector of (3).

  Examples of pharmaceutically acceptable carriers include excipients such as sucrose and starch, binders such as cellulose and methylcellulose, disintegrants such as starch and carboxymethylcellulose, lubricants such as magnesium stearate and aerosil, citric acid, Fragrances such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspensions such as methylcellulose and polyvinylpyrrolide, dispersants such as surfactants, water, Although diluents, such as physiological saline, base wax, etc. are mentioned, it is not limited to them.

  Furthermore, when the vaccine of the present invention is the expression vector of (3) above, the vaccine of the present invention can further contain a nucleic acid introduction reagent in order to promote introduction of the expression vector into cells. When a viral vector is used as an expression vector, retronectin, fibronectin, polybrene or the like can be used as a gene introduction reagent. When a plasmid vector is used as an expression vector, lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, poly (ethyleneimine) (PEI), etc. Cationic lipids can be used.

  In order to enhance the immunogenicity of the peptide encoded by the peptide of (1) or (2) or the expression vector of (3), the vaccine of the present invention preferably further comprises a carrier protein. A carrier protein is a substance that imparts immunogenicity by binding to a molecule that is not immunogenic due to its low molecular weight, and is known in the art. Preferred examples of the carrier protein include bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), squash hemocyanin (KLH), thyroglobulin (TG), immunoglobulin and the like. A particularly preferred carrier protein is mussel hemocyanin (KLH). In the case of the expression vector (3) above, the nucleotide encoding the carrier protein may be linked to the nucleotide encoding the peptide (1) or (2).

  The vaccine of the present invention preferably further contains an adjuvant that is pharmaceutically acceptable and compatible with the active ingredient. Adjuvants are generally substances that non-specifically enhance the host immune response, and a number of different adjuvants are known in the art. Examples of adjuvants include, but are not limited to: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1′-2′-dipalmitoyl) -Sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE), Quill A (registered trademark), lysolecithin, saponin derivative, pluronic polyol, Montanide ISA-50 (Seppic, Paris, France), Bayol (registered trademark) ) Fine Markol (registered trademark).

  The vaccine of the present invention can be administered to mammals orally or parenterally. Since polypeptides and expression vectors can be degraded in the stomach, they are preferably administered parenterally. Examples of the preparation suitable for oral administration include liquids, capsules, sachets, tablets, suspensions, emulsions and the like. Formulations suitable for parenteral administration (eg, subcutaneous injection, intramuscular injection, local infusion, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. Alternatively, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

The content of the active ingredient in the pharmaceutical composition is usually about 0.1 to 100% by weight, preferably about 1 to 99% by weight, more preferably about 10 to 90% by weight of the whole pharmaceutical composition.
The dosage of the vaccine of the present invention varies depending on the subject to be administered, the administration method, the dosage form, etc., but in the case where the active ingredient is the peptide of (1) or (2), the polypeptide is usually administered once per adult. In the range of 1 μg to 1000 μg per dose, preferably in the range of 20 μg to 100 μg, usually 2 to 3 times over a period of 4 to 12 weeks. When the antibody titer decreases, it is further administered once each time. When the active ingredient is the expression vector of (3) above, the expression vector per adult is usually in the range of 1 μg to 1000 μg, preferably in the range of 20 μg to 100 μg, usually for 4 to 12 weeks. Administer 2 to 3 times. If the antibody titer decreases, add 1 dose each time.

  By administering the vaccine of the present invention to a mammal, T cell differentiation is shifted from Th1 to Th2, and a decrease in blood C peptide level and T cell infiltration into islets are suppressed, resulting in type 1 diabetes and type 1 Preventive or therapeutic effects on diseases associated with diabetes are exerted.

  The present invention also provides a kit comprising one or more containers including one or more components of the vaccine of the present invention. By using the kit of the present invention, type 1 diabetes and its related diseases can be prevented, or the symptoms can be treated or alleviated.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

Animal research and immunity
Nine-week-old female NOD / ShiLtJ mice (n = 30) were purchased from Charles River (Japan), raised in a temperature and light cycle controlled facility, and freely fed food and water. A mouse was diagnosed as a diabetic mouse when the continuous glucose concentration for 2 consecutive days by a glucometer (Sanwa Kagaku Kenkyusho, Japan) was higher than 250 ng / dl. Prior to immunization, the peptide solution was mixed with an equal volume of complete / incomplete Freund's adjuvant (CFA / IFA; Wako, Japan). In the vaccine group, NOD mice (n = 6) were injected subcutaneously with 50 μg of GFAP peptide (C4) at 0, 2, 4 and 42 weeks. In the KLH control group, an equal amount of KLH mixed with an equal amount of Freund's adjuvant was injected. In the physiological saline control group, an equal amount of physiological saline was injected. Serum was collected from the tail vein and the antibody titer against the immunizing peptide was measured by ELISA.

Vaccine design and synthesis Based on high antigenicity analysis, the inventors derived four peptides from mouse GFAP protein: C1 peptide (79-87aa; SEQ ID NO: 5), C2 peptide (102-110aa; SEQ ID NO: 6), C3 peptide (276-284aa; SEQ ID NO: 8) and C4 peptide (414-427aa; SEQ ID NO: 10) were selected. BSA was conjugated to the N-terminus of the candidate antigen sequence, and the synthetic peptide was purified by reverse phase HPLC (Peptide Institute Inc., Osaka, Japan.) (> 98% purity).

ELISA assay In the antibody titer assay, ELISA plates (MaxiSorp Nunc, Thermo Fisher Scientific KK, Japan) were used with 5 μg / ml candidate GFAP peptides (4 types above), 5 μg recombinant GFAP protein (Cusabio, China) or 2 μg recombination. Coat with GAD65 protein (Abnova Taiwan) in carbonate buffer at 4 ° C. overnight. The peptide was previously conjugated to the carrier protein BSA (Peptide Institute Inc., Japan). After blocking all wells with PBS containing 3% skim milk, the serum was diluted 100 to 325,000 times with blocking buffer. After incubating the plate with serum (overnight at 4 ° C.) and washing, the plate was incubated with mouse IgG specific HRP-conjugated antibody (GE Healthcare. UK) for 2 hours at room temperature. In the IgG subclass determination assay, anti-mouse Ig subclass-specific HRP-conjugated antibodies (IgG1, IgG2b and IgG2c) were used. After washing the plate with PBS, color was developed with 3,3 ′, 5,5′-tetramethylbenzidine (TMB; Sigma Aldrich), a peroxidase chromogenic substrate, and the reaction was terminated with O.5N sulfuric acid. Absorbance at 450 nm was measured using a microplate reader (Bio-Rad Inc., Japan). The half-maximum antibody titer was determined according to the maximum value within the dilution range of each sample.

Peptide assays and histological analysis
For the C peptide assay, 100 μl Working Strength Conjugate was mixed with 10 μl serum from a 16 hour fasted NOD mouse in each well (96 well plate Mouse C peptide ELISA, ALPCO, US) for 2 hours at room temperature. After incubation with TMB substrate and stop solution, absorbance at 450 nm was measured with a microplate reader (Bio-Rad Inc., Japan). For histological analysis, the pancreas was collected, fixed overnight in 4% formalin, embedded in paraffin, sliced into 4 μm sections, and stained with hematoxylin and eosin (HE). For each mouse (n = 3), at least 35 islets were analyzed per mouse. Staining was analyzed according to the method described in Weaver, DJ, et al, Journal of immunology (Baltimore, Md .: 1950) 167, 586-592 (2001). Normal islet: no T cell infiltration, before isletitis: T cell infiltration into islet, isletitis: almost 100% of T cell infiltration into islet

ELISPOT assay
A 96-well ELISPOT plate (Millipore, Japan) was coated with anti-mouse IFN-α capture antibody or anti-mouse IL-4 capture antibody at 4 ° C. overnight. After incubation, the plates were washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked with PBS containing 1% BSA and 5% sucrose. Next, a mouse-derived spleen cell suspension was added to the plate (10 ⁶ cells / well), and GFAP (C3 or C4) peptide, GFAP protein, GAD65 protein, KLH and PHA were added at 10 μg / ml at 37 ° C. for 48 hours. I was stimulated. After incubation, the plate was washed with PBS-T. Wells were incubated overnight at 4 ° C. with biotinylated anti-mouse IFN-α antibody or biotinylated anti-mouse IL-4 antibody and washed with PBS-T. Streptavidin-AP was added to each well and incubated for 2 hours at room temperature. After washing with PBS-T, the plate was incubated with BCIP / NBT solution for 30 minutes at room temperature. Finally, the plate was washed with water, air dried at room temperature, and the number of colored spots was counted using a dissecting microscope (Leica LMD6500, Germany).

Statistical analysis All data were expressed as mean ± standard error. Differences between the two groups were verified using the unpaired 2-tailed Student's t-test. Correlation between autoantibody titer and C-peptide was verified using unpaired 2-tailed Pearson correlation analysis. Data sets involving more than one group were verified with a Tukey's post-hoc test using Prism GraphPad version 5.01 (GraphPad Software Inc.). Statistical differences were considered significant when P <0.05.

Example 1 Relationship between GFAP and C peptide in NOD mice
NOD mice are known as type 1 diabetes model mice. The inventors evaluated the onset of type 1 diabetes in female NOD mice (n = 30) from 9 weeks of age (FIG. 1A). NOD mice were determined to develop diabetes when the blood glucose level at any time was greater than 250 mg / ml for 2 consecutive days. As a result, the onset of diabetes in NOD mice was confirmed from 18 weeks of age. In addition, the inventors confirmed that almost half or more of the mice were diabetic at 30 weeks of age. So far, it has been reported that antibodies to the self-antigen GFAP increase in NOD mice in an age-dependent manner, so the inventors focused on anti-GFAP antibodies and produced anti-GFAP antibodies in NOD mice from 9 weeks of age. Were evaluated and compared to antibody production against the known autoantigen GAD65 associated with type 1 diabetes. As a result, anti-GFAP antibody increased in a time-dependent manner (FIG. 2A). Similarly, anti-GAD65 antibody also increased in a time-dependent manner (FIG. 2B). However, the fold increase in anti-GFAP antibodies at 21 and 25 weeks of age was not correlated with glucose levels at 27 weeks of age (FIG. 3A). Similarly, the fold increase of anti-GAD65 antibodies at 21 and 25 weeks of age was not correlated with glucose levels at 27 weeks of age (FIG. 3B).
Next, the inventors examined the relationship between anti-GFAP antibody, anti-GAD65 antibody and C peptide secretion reflecting β-cell function. As a result, when tested in all 25-week-old NOD mice without distinguishing between diabetic and non-diabetic groups, production of anti-GFAP and anti-GAD65 antibodies and fasting C-peptide secretion did not correlate (FIG. 4A, B). Furthermore, all the 25-week-old NOD mice were divided into a diabetic group (DM; glucose level greater than 250 mg / ml) and a non-diabetic group (non-DM; glucose level below 250 mg / ml), and verified. In the diabetic group, production of anti-GFAP antibody and anti-GAD65 antibody and fasting C-peptide secretion showed an inverse correlation (FIGS. 4C and D). On the other hand, in the non-diabetic group, production of anti-GFAP antibody and anti-GAD65 antibody did not correlate with fasting C-peptide secretion (FIGS. 4E and F). These results indicate that anti-GFAP and anti-GAD65 antibodies may be involved in the reduction of C peptide secretion in diabetic mice.

Example 2 Prediction of type 1 diabetes by detection of anti-GFAP antibody
In order to evaluate whether anti-GFAP antibody and anti-GAD65 antibody can be used as predictive markers for diabetes in NOD mice, the antibodies were positive at 17, 21 and 25 weeks of age (compared to the antibody titers of 9-week-old NOD mice) Thus, it was investigated whether or not the mice whose antibody titers at 17, 21, and 25 weeks of age were more than twice developed diabetes at 30 weeks of age. A mouse was diagnosed as a diabetic mouse when the continuous glucose concentration for 2 consecutive days by a glucometer (Sanwa Kagaku Kenkyusho, Japan) was higher than 250 ng / dl. As a result, 30% of mice determined to be anti-GFAP antibody positive at 17 weeks of age and about 70% of NOD mice determined to be anti-GFAP antibody positive at 21 or 25 weeks of age developed diabetes (FIG. 5A). ). These results indicate that anti-GFAP antibody is a sensitive marker of the onset of diabetes in NOD mice. Similarly, NOD mice that were positive for anti-GAD65 antibody were examined, about 20% of mice that were determined to be anti-GAD65 antibody positive at 17 weeks of age, and mice that were determined to be anti-GAD65 antibody positive at 25 weeks of age. Of these, 43% developed diabetes (FIG. 5B). Furthermore, about 35% of NOD mice judged to be positive for anti-GFAP antibody and anti-GAD65 antibody at 21 or 25 weeks of age developed diabetes (FIG. 5C). This result shows that the anti-GFAP antibody and the anti-GAD65 antibody were consistent in correlating with the onset of type 1 diabetes, but the anti-GFAP antibody is more sensitive. In addition, the relationship between the onset of type 1 diabetes and anti-GFAP antibody and anti-GAD65 antibody-negative mice weakened in a time-dependent manner (FIG. 5D). These results indicate that anti-GFAP antibody and anti-GAD65 antibody are reliable predictive markers for the onset of type 1 diabetes in NOD mice.

Example 3 Selection and Screening of Antigenic Sequence of GFAP Vaccine The inventors further analyzed the anti-GFAP antibody of NOD mouse, and based on the three-dimensional structure prediction of GFAP, four candidate peptides (C1 Peptide, C2 peptide, C3 peptide, C4 peptide) were selected. In 28-week-old NOD mice, anti-GFAP antibody mainly bound to C3 peptide (FIG. 6A). This result suggests that the anti-GFAP antibody mainly binds to the C3 peptide area. In addition, the inventors examined an IgG subclass of anti-GFAP antibodies that can bind to C3 peptides using an ELISA assay. Similar to the results of the anti-GFAP antibody binding to the full-length GFAP protein of NOD mice (FIG. 1B), the ratio of IgG2b of the anti-GFAP antibody binding to the C3 peptide was larger than IgG1 (dilution 1: 100) (FIG. 6B). ). This result indicates that an antibody using C3 peptide as an antigen is associated with T cells differentiated into Th1 type, suggesting an association with the onset of type 1 diabetes.

Example 4 Modulation of Type 1 Diabetes in NOD Mice with GFAP Vaccines The inventors designed a therapeutic GFAP vaccine to induce immune tolerance against NOD mice. Type 1 diabetes is a T cell-dependent tissue-specific autoimmune disease, but it cannot be denied that pancreatic β-cells may be damaged by antibodies produced from B cells, so it is elevated in NOD mice The C4 peptide was selected avoiding the antigen sequence of the C3 peptide. Furthermore, the inventors designed a peptide vaccine (GFAP vaccine) conjugated with KLH that shifts the immune response in the Th2 direction to the C4 peptide. The vaccine was administered 3 times every 2 weeks to 9 week old female NOD mice (n = 6). Each mouse received 50 μg of peptide in combination with Freund's adjuvant. Antibody production against the GFAP vaccine was assessed by ELISA titer assay. Induced antibodies markedly increased in NOD mice immunized with GFAP vaccine, but no increase in anti-GFAP antibody was observed in mice administered KLH vaccine or saline. Furthermore, the increased antibody titer reached a maximum at 30 weeks of age (FIG. 7A). In order not to attenuate the antibody titer, the NOD mouse was reboosted with the GFAP vaccine at 42 weeks of age. As a result, the antibody titer increased again.
In order to verify the effect of GFAP vaccine on the development of type 1 diabetes in NOD mice, the inventors developed diabetes onset in groups of mice treated with saline, GFAP vaccine or KLH vaccine (glucose levels from 250 mg / ml). Evaluated). As shown in FIG. 7B, most of the mice treated with physiological saline developed diabetes, whereas GFAP vaccine or KLH vaccine completely suppressed the development of diabetes in 52-week-old NOD mice. Furthermore, the fasting C-peptide level in the serum of 37-week-old NOD mice was significantly lower than that of mice immunized with GFAP vaccine or KLH vaccine (FIG. 8A). These results indicate that GFAP vaccine or KLH vaccine effectively delays the onset of type 1 diabetes in NOD mice. In addition, in order to verify whether the GFAP vaccine can effectively suppress isletitis, the inventors conducted histological analysis of the islets. NOD mice administered with the GFAP vaccine significantly suppressed T cell infiltration compared to mice administered with saline and KLH vaccine (FIG. 8B). These results suggest that the GFAP vaccine significantly improves diabetes symptoms in NOD mice by suppressing the infiltration of T cells into the islets. Interestingly, the 30-week-old NOD mice that received the KLH vaccine had significantly greater infiltration of T cells than the mice that received the GFAP vaccine, but the KLH and GFAP vaccine groups There was no significant difference in diabetes symptoms.

Example 5 Shift of immune response from Th1 bias to Th2 bias by GFAP vaccine
T cell responses in type 1 diabetes and other autoimmune diseases are regulated by Th1 lymphocytes. Therefore, the inventors evaluated the T cell activity of NOD mice that developed type 1 diabetes and NOD mice that did not develop type 1 diabetes using the ELISPOT assay (FIG. 9). Stimulation with C3 peptide (276-284aa), recombinant GFAP (full length), and recombinant GAD65 (full length) each significantly increased spleen cells producing IFN-γ from IL-4. That is, since significant activation of IFN-γ was observed, it was found that these stimuli promoted activation of Th1 lymphocytes, which are T cell responses observed in type 1 diabetes. Next, in order to confirm the binding site of the antibody induced by the GFAP vaccine, the inventors were administered the GFAP vaccine using an ELISA assay with immobilized C1, P2, C3 or C4 peptides. Anti-GFAP antibodies derived from NOD mice were evaluated. In mice immunized with GFAP vaccine, the antibody binding to the C4 peptide was significantly increased compared to antibodies binding to the other three peptides (FIG. 8C). Interestingly, despite the immunization with the vaccine using the C4 peptide, antibodies against the surrounding antigens C1, C2, and C3 peptides also increased (antibody spreading), administered KLH vaccine or saline In the NOD mice, the production of antibodies against C3 peptide was much greater than that against C1, P2 and C4 peptides. Furthermore, autoantibodies to C3 peptide were significantly reduced in mice immunized with KLH vaccine than in the saline group. These results suggested that the suppression of diabetes by KLH vaccine was due to a decrease in cytotoxic antibodies (autoantibodies against C3 peptide). Furthermore, in order to verify whether the GFAP vaccine shifts the T cell response in NOD mice, the inventors assessed T cell differentiation using IgG subclass ELISA assay and ELISPOT assay. In the IgG subclass analysis, GFAP vaccine induced IgG1 production stronger than IgG2b and IgG2c production (FIG. 8D). When the β cells of the pancreas were confirmed pathologically, the damage of β cells of insulitis or pre-insulitis was reduced in the GFAP vaccine group (FIGS. 8B and 10A). As shown in FIG. 10B, stimulation with KLH induced IL-4 and IFN-γ production from the spleen cells of mice immunized with GFAP vaccine, resulting in significant production of IL-4 in the ELISPOT assay. . GFAP peptide (C4) did not induce a T cell response compared to the unstimulated group. In addition, NOD mice immunized with KLH vaccine significantly induced IL-4 and IFN-γ production after KLH stimulation (FIG. 10C). These results suggest that the GFAP vaccine shifts the T cell response from Th1 bias to a more harmless and protective Th2 bias. Overall, these results suggest that the GFAP vaccine significantly delays the onset of type 1 diabetes and suppresses C peptide loss by shifting the T cell response from Th1 to Th2 in spleen cells. It was done.

  By detecting an autoantibody that recognizes a specific amino acid sequence of GFAP from a sample of a subject, it can be accurately determined whether the subject develops type 1 diabetes or will develop in the future. In addition, by using a specific amino acid sequence of GFAP as a vaccine, it suppresses T cell reactivity, and induces T cell differentiation from Th1 to Th2 to induce type 1 diabetes and type 1 diabetes related diseases. Can prevent or treat the onset.

Claims (8)

A method for testing type 1 diabetes, comprising measuring the following autoantibody (1) or (2) from a sample derived from a subject:
(1) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or an autoantibody against a peptide consisting of the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal, and (2) an amino acid sequence shown in SEQ ID NO: 2. Or an autoantibody against a peptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal.   The test method according to claim 1, wherein the sample is plasma or serum.   The method according to claim 1 or 2, wherein autoantibodies are measured by an immunological method. A test kit for type 1 diabetes comprising the following peptide (1) or (2):
(1) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a peptide consisting of the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal, and (2) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2. Alternatively, a peptide comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence corresponding to SEQ ID NO: 2 in a non-human mammal. A vaccine for preventing or treating type 1 diabetes or a disease associated with type 1 diabetes, comprising any of the following substances (1) to (3):
(1) A peptide comprising the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal;
(2) An amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4 or the amino acid sequence corresponding to SEQ ID NO: 4 in a non-human mammal And (3) an expression vector capable of expressing the peptide of (1) or (2) above.   6. A vaccine according to claim 5, comprising a carrier protein.   The vaccine according to claim 6, wherein the carrier protein is squash hemocyanin.   The vaccine according to any one of claims 5 to 7, comprising an adjuvant.