JP2021038159A - RAGE-derived peptides and their use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vaccine for prevention or treatment of a disease which develops or progresses due to enhancement of action of AGEs-RAGE system due to accumulation of AGEs or enhancement of expression and function of RAGE. The present invention is a vaccine for preventing or treating AGEs-RAGE-related diseases, which comprises the following peptides (a) or (b). One or several amino acids have been substituted, deleted, inserted and / or added in (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or (b) the amino acid sequence represented by SEQ ID NO: 1. Peptide consisting of amino acid sequence and having the same immunogenicity as the peptide of (a) above [Selection diagram] None

Description

The present invention relates to a partial peptide derived from RAGE and a vaccine containing the partial peptide.

Diabetic kidney disease is a general term for renal disorders caused by diabetes, and when it progresses in a chronic course, it causes irreversible end-stage renal disease, but it is a poor factor for cardiovascular prognosis and life prognosis even in the early stage.

For diabetic kidney disease, a multifaceted approach that combines blood glucose control with hypoglycemic agents and antihypertensive treatment mainly with renin-angiotensin system inhibitors is a pillar of prevention and treatment strategies. Globally, the onset and progression of diabetic kidney disease have not been sufficiently controlled. Therefore, it is considered that it is an urgent medical issue to develop and establish a new preventive and therapeutic method for diabetic kidney disease, which is important not only for improving the prognosis of individual patients but also for medical economics. Be done.

Long-term hyperglycemic conditions have caused metabolic and hemodynamic abnormalities involving advanced glycation end products (AGEs) and their receptors, RAGE (receptor for AGEs) (AGEs-RAGE system). It is known that it is used (Non-Patent Document 1). Therefore, it has been thought that suppressing the action of AGEs-RAGE will lead to the development of therapeutic strategies specific to diabetic kidney disease.
However, AGEs are a general term for structures in which proteins undergo non-enzymatic glycation modification, and since they are a heterogeneous population, they are difficult to target for treatment using active immunity (vaccine).
On the other hand, RAGE has a known three-dimensional structure, and AGEs bind to the extracellular domain and are involved in pathogenesis, so it was considered to be suitable as a target for active immunotherapy. So far, therapeutic methods targeting RAGE have been neutralizing antibody (Non-Patent Document 2), DNA aptamer (Non-Patent Document 3), antagonist (FPS-ZM1: Non-Patent Document 4, Azeliragon (TTP448): Non-Patent Document). Although both Documents 5 and 5 are being developed as therapeutic agents for Alzheimer-type dementia), no drug has been approved as a therapeutic agent for diabetic kidney disease to date.

Diabetic kidney disease progresses in a chronic course and has few subjective symptoms at an early stage, so that treatment adherence tends to decrease, often leading to treatment discontinuation. In this regard, by nature, if a vaccine can induce the production of an effective antibody against a target protein, it can be expected that the therapeutic effect will continue for a long period of time with a small number of doses, which can contribute to the improvement of therapeutic adherence. Therefore, it is considered to be an effective preventive treatment strategy.
So far, diabetic nephropathy therapeutic vaccines targeting MCP1 (Non-Patent Document 6), AGEs (Non-Patent Document 7), TGF-β (Non-Patent Document 8), and angiotensin receptor (Non-Patent Document 9) Development has been attempted, and animal experiments have suggested an effect of improving diabetic kidney disease.
However, the development of therapeutic vaccines targeting RAGE has not been reported to date.

Diseases that develop or progress due to the enhancement of the action of the AGEs-RAGE system due to the accumulation of AGEs or the enhancement of the expression and function of RAGE are wide-ranging other than diabetic kidney disease (Non-Patent Documents 11 to 18).
Therefore, it is considered to be an important solution in the art to develop a drug that suppresses a disease that develops or progresses due to an enhanced action of the AGEs-RAGE system.

Tanji et al., J Am Soc Nephrol. 11: 1656-1666 2000. Flyvbjerg et al., Diabetes 53: 166-172 2004. Matsui et al., Diabetes 66: 1683-1695 2017. Deane et al., J Clin Invest. 122: 1377-1392 2012. Galasko et al., Neurology 82: 1536-1542 2014. Celec et al., Hum Gene Ther. 23: 158-166 2012. Mashitah et al., Diabetes Metab Syndr Obes. 8: 347-355 2015. Zhang et al., Immunotherapy. 8: 1045-1057 2016. Ding et al., J Mol Med (Berl). 94: 207-218 2016. Yamagishi et al., Oxid Med Cell Longev. 3: 101-108 2010. Toth et al., Diabetes. 57: 1002-1017 2008. Barile et al., Invest Ophthalmol Vis Sci. 46: 2916-2924 2005. Soro-Paavonen et al., Diabetes 57: 2461-2469 2008. Li et al., PLoS One 8: e76025 2013 Koyama et al., J Card Fail. 13: 199-206 2007. Kanauchi et al., Diabetes Care. 24: 1620-1623 2001. Yan et al., Nature 382: 685-691 1996. Caspar-Bell et al., Mol Cell Biochem. 414: 171-178 2016. Chen et al., Adv Drug Deliv Rev. 65: 1357-69 2013.

In view of the above circumstances, it is an object of the present invention to provide a vaccine for the prevention or treatment of diseases that develop or progress due to the enhancement of the action of the AGEs-RAGE system due to the accumulation of AGEs or the enhancement of the expression and function of RAGE. And.

In order to solve the above problems, the present inventors prepared a vaccine containing a partial sequence of a carrier protein (for example, KLH) and RAGE, and as a result of diligent studies on its effect, the vaccine was found to be streptozotocin-induced diabetes mellitus. It has been found that diabetic kidney disease is alleviated not only in mice but also in db / db mice that develop diabetes due to gene mutation.

That is, the present invention is the following (1) to (5).
(1) A vaccine for preventing or treating AGEs-RAGE-related diseases, which comprises the following peptides (a) or (b).
One or several amino acids have been substituted, deleted, inserted and / or added in (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or (b) the amino acid sequence represented by SEQ ID NO: 1. A peptide consisting of an amino acid sequence and having the same immunogenicity as the peptide of (a) above (2) The carrier protein is fused to the peptides of (a) and (b) above. The vaccine according to (1) above.
(3) The vaccine according to (1) or (2) above, which comprises the peptide of (a) or (b) or a vector capable of expressing a fusion of these peptides and a carrier protein.
(4) The isolated peptide of (a) or (b) below.
One or several amino acids have been substituted, deleted, inserted and / or added in (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or (b) the amino acid sequence represented by SEQ ID NO: 1. A peptide consisting of an amino acid sequence and having the same immunogenicity as the peptide of (a) above (5) A nucleic acid encoding the peptide according to (4) above.

INDUSTRIAL APPLICABILITY According to the present invention, the prevention and / or treatment of diseases that develop or progress due to the enhancement of the action of the AGEs-RAGE system due to the accumulation of AGEs or the expression and function of RAGE, for example, diseases such as diabetic kidney disease. Vaccine for is provided.

RAGE peptide (SEQ ID NO: 1) specific IgG antibody titer. The results of measuring the serum IgG antibody titer against the RAGE peptide after subcutaneous injection of the RAGE peptide-KLH fusion and various adjuvants (FCA / FIA, Alum and polyIC) into mice are shown. **: p <0.01 vs Alum, †: p <0.05 vs polyIC, Fisher's PLSD (constrained minimum significant difference test), n = 4 / group. Changes in RAGE peptide-specific IgG antibody titer over time. The results of evaluating the time course of RAGE peptide-specific IgG antibody titer after subcutaneous injection of RAGE peptide-KLH fusion and Freund's adjuvant to mice are shown. Examination of the effect of the vaccine of the present invention (RAGE peptide-KLH) on blood glucose level in type 1 diabetes model mice. Diabetes was induced by subcutaneous injection of RAGE-KLH with Freund's adjuvant (vaccine) or KLH with adjuvant (vehicle) into mice and then streptozotocin at 10 weeks of age. The measurement results of the blood glucose level 2 weeks or 8 weeks after the administration of streptozotocin are shown. n = 10 / group. Examination of the effect of the vaccine of the present invention on albuminuria in type 1 diabetes model mice. The measurement result of the urinary albumin excretion 8 weeks after the administration of streptozotocin in the type 1 diabetes model mouse (same as the explanation of FIG. 3) is shown. The value is shown as the amount of albumin (μg) per 1 mg of creatinine in urine. *: P <0.05 vs vehicle (unpaired t-test), n = 10 / group. Examination of the effect of the vaccine of the present invention on glomerular swelling and mesangial matrix expansion in type 1 diabetes model mice. The observation result of the renal tissue 12 weeks after the administration of streptozotocin in the type 1 diabetes model mouse (same as the explanation of FIG. 3) is shown. Images of kidney tissue are the result of PAS staining. The graph on the left shows the glomerular volume, and the graph on the right shows the ratio of the mesangial substrate area to the glomerular area. **: p <0.01 vs vehicle (according to unpaired t-test), n = 7-9 / group. Examination of the effect of the vaccine of the present invention on glomerular basement membrane thickening in type 1 diabetes model mice. The results of electron microscopic observation of renal tissue 12 weeks after streptozotocin administration in type 1 diabetes model mice (same as the explanation in FIG. 3) are shown. The figure on the left is the measurement result of the thickness of the glomerular basement membrane, and the figure on the right is the observation image of the kidney with an electron microscope. **: p <0.01 vs vehicle (unpaired t-test), n = 4 / group. Examination of the effect of the vaccine of the present invention on albuminuria in type 2 diabetes model mice. Male db / db mice were injected subcutaneously with RAGE-KLH with Freund's adjuvant (vaccine) or KLH with adjuvant (vehicle). The measurement results of urinary albumin excretion immediately before immunization (at 10 weeks of age) and 14 weeks after the final immunization are shown. *: p <0.05 vs vehicle (unpaired t-test), n = 6 / group. Examination of the effect of the vaccine of the present invention on glomerular swelling and mesangial matrix expansion in type 2 diabetes model mice. Male db / db mice were injected subcutaneously with RAGE-KLH with Freund's adjuvant (vaccine) or KLH with adjuvant (vehicle). Twenty weeks after final immunization, mouse kidneys were evaluated by PAS staining. The graph on the left shows the glomerular volume, and the graph on the right shows the ratio of the mesangial substrate area to the glomerular area. *: p <0.05 vs vehicle, **: p <0.01 vs vehicle (according to unpaired t-test), n = 5 / group.

Hereinafter, modes for carrying out the present invention will be described.
The first embodiment is a vaccine containing a peptide consisting of a partial amino acid sequence of RAGE or a vector capable of expressing the peptide, which is an AGEs-RAGE system induced by the expression and functional enhancement of AGEs and RAGE accumulated in the body. It is a vaccine for the prevention or treatment of diseases caused by enhanced action (hereinafter, also referred to as "AGEs-RAGE related diseases"). AGEs are recognized by RAGE, which is a cell surface receptor, and are considered to induce oxidative stress and inflammatory reaction, and to induce angiopathy (Non-Patent Document 10). The AGEs-RAGE-related diseases are not particularly limited, but are, for example, diabetic kidney disease (DKD), diabetic neuropathy (Non-Patent Document 11), and diabetic nephropathy (Non-Patent Document 12). Diabetic diseases such as arteriosclerosis (Non-Patent Document 13), cerebrovascular disease (Non-Patent Document 14), heart failure (Non-Patent Document 15), coronary artery disease (Non-Patent Document 16), Alzheimer's disease (Non-Patent Document 17) ) And hyperthyroidism (Non-Patent Document 18).

More specifically, in the first embodiment, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (that is, CKGAPKK) and a peptide consisting substantially the same as the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 Alternatively, it is a vaccine for preventing or treating AGEs-RAGE-related disorders (hereinafter, also referred to as "the vaccine of the present embodiment") containing a vector capable of expressing these peptides.
That is, the first embodiment is a vaccine for preventing or treating AGEs-RAGE-related diseases, which comprises the following peptides (a) or (b).
One or several amino acids have been substituted, deleted, inserted and / or added in (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or (b) the amino acid sequence represented by SEQ ID NO: 1. A peptide consisting of an amino acid sequence and having the same immunogenicity as the peptide of (a) above. Hereinafter, the peptides of (a) and (b) above are also referred to as "peptides of the present embodiment".

Here, the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is a peptide obtained by separating a part of human RAGE represented by SEQ ID NO: 3, and is the 38th to 44th peptides in the full-length amino acid sequence of RAGE. Corresponds to the amino acid sequence portion. Further, "a peptide substantially the same as the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1" is one or several (preferably 1, 2 or 3) in the amino acid sequence represented by SEQ ID NO: 1. A peptide consisting of an amino acid sequence in which (1) amino acids are substituted, deleted, inserted and / or added, and has the same immunogenicity as the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (for example, A peptide that induces an immune response specific to RAGE).

The amino acid "substitution" is preferably, for example, a conservative amino acid substitution. Here, "conservative amino acid substitution" is to replace an arbitrary amino acid with another amino acid having a side chain having the same physicochemical properties as the side chain of the amino acid. A group of amino acids having side chains with similar physicochemical properties can be easily identified by those skilled in the art. The group of such amino acids includes, but is not limited to, for example, a group of amino acids having a basic side chain (eg, lysine, arginine, histidine, etc.), a group of amino acids having an acidic side chain (eg, aspartic acid, glutamic acid). ), A group of amino acids having a neutral side chain (eg, glycine, aspartin, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and the like. .. Also, a group of amino acids with polar side chains (eg, glycine, asparagine, glutamine, serine, trionine, tyrosine, cysteine), a group of amino acids with non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, Phenylalanine, methionine, tryptophan) and the like.

The C-terminus of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is usually a carboxyl group (-COOH) or a carboxylate (-COO). In addition to (-), the carboxyl group _{may be chemically modified with an amide (-CONH 2} ), an ester (-COOR), or the like. Here, as R in the ester, an alkyl group having 1 to 6 carbon atoms (for example, methyl, ethyl, n-propyl, isopropyl or n-butyl) and a cycloalkyl group having 3 to 8 carbon atoms (for example, cyclopentyl or Cyclohexyl), aryl groups with 1 to 6 carbon atoms (eg, phenyl or α-naphthyl), phenyl-C1-2 alkyl groups (eg, benzyl or phenethyl), α-naphthyl-C1-2 alkyl groups (eg, α- Naftylmethyl) and the like. In addition to the C-terminal, peptides having a carboxyl group in the peptide chain and having the carboxyl group amidated or esterified are also included in the peptide of the present embodiment. Examples of the ester in this case include the above-mentioned esters.

Further, the N-terminal of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and the peptide consisting substantially the same as the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is usually an amino group (-NH ₂ ). The amino group may be chemically modified with an acyl group having 1 to 6 carbon atoms such as a formyl group or an acetyl group. In addition, the glutamil group generated by cleaving the N-terminal side in vivo is pyroglutamine-oxidized, and the substituent on the side chain of the amino acid in the molecule (for example, -OH, -SH, amino group, imidazole group, indol group). Alternatively, the guanidino group, etc.) may be chemically modified with a suitable functional group (eg, formyl group, methyl group, acetyl group, etc.), or a sugar chain may be attached.

The peptide of the present embodiment can be produced by a method well known in the art, for example, a genetic engineering method, or a chemical peptide synthesis method (solid phase method or liquid phase method).
When chemically synthesizing a peptide, a part or amino acid of the peptide constituting the peptide of the present embodiment is condensed with a residual part, and when the product has a protecting group, the protecting group is removed to obtain the desired substance. Peptides can be produced. After the synthetic reaction, the peptide of the present embodiment can be isolated and purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, high performance liquid chromatography, and recrystallization.
When the peptide of the present embodiment is genetically engineered, a vector (expression vector) containing a DNA encoding the peptide of the present embodiment (for example, a DNA consisting of the nucleic acid sequence represented by SEQ ID NO: 2) is used. The host cell may be transformed with, and the obtained transformed cell may be cultured to isolate and purify the target peptide (the peptide of the present embodiment) from the culture.

Furthermore, the peptide contained in the vaccine of the present embodiment may be a fusion bound to a carrier protein. The "carrier protein" is a protein that functions to enhance the immunogenicity of the peptide, and a person skilled in the art can select a desired protein, such as scallop hemocyanin (KLH) and egg white albumin (KLH). OVA), rabbit serum albumin (RSA), bovine serum albumin (BSA), thyroglobulin (TG), immunoglobulin, tetanus toxoid, diphtheria toxoid, diphtheria toxin variant CRM197, pneumoniae surface antigen A (PspA), pneumoniae surface antigen C (PspC), pneumococcal histidine protein A (PhpA), hepatitis B virus surface antigen (HBs), hepatitis B virus core antigen (HBc), hepatitis E virus capsid protein (eg ORF (open reading frame) 2), etc. ), HIV-1 enveloped glycoprotein (eg gp41, gp120 and gp160), hepatitis C virus enveloped glycoprotein (eg E1E2, NS3, NS4 and NS5B), influenza M2 protein, influenza hemagglutinin protein (HA), malaria protozoa Sporozoite surface protein, tuberculosis Ag85 complex protein, tuberculosis heat shock protein (eg Hsp65, Hsp70), tuberculosis low molecular weight secretory protein (eg, early secretory antigen target (ESAT) -6, culture filtration protein (CFP) -10) , Human papillomavirus L1 capsid protein, human papillomavirus 16 neoplastic antigens (eg E6, E7), Niseria heparin binding antigen (NHBA), meningitis H factor binding protein (fHbp), meningitis surface adhesin A ( NadA), choleratoxin B subunit (CTB), nontoxic mutant heat-resistant Escherichia coli toxin (LTB), botulinum toxin heavy chain C-terminal domain (BoHc), norovirus capsid protein (eg VP1), rotavirus antigen (eg VP2, VP4) , VP6, VP7) and so on. Of these, the carrier protein is preferably mussel hemocyanin (KLH).
The peptide of the present embodiment and the carrier protein may be directly bound or may be bound via a linker or the like. The arrangement of the peptide and the carrier protein of the present embodiment may be any arrangement, and for example, a configuration in which the carrier protein is arranged at the N-terminal or the C-terminal of the peptide of the present embodiment can be exemplified. The number of the peptides contained in the fusion of the peptide of the present embodiment and the carrier protein may be one or a plurality.

When the peptide of the present embodiment and the carrier protein are directly bonded, various condensing agents such as dimaleimide compound, maleimide active compound, carbodiimide compound, diazonium compound, dialdehyde compound, diisocyanate compound and the like are used by a coupling reaction. Can be combined. These coupling reactions can be carried out based on techniques well known to those skilled in the art. In addition, when the peptide of the present embodiment and the carrier protein are bound via a linker peptide, it can be easily carried out using a linker peptide known in the art. Examples of the linker peptide include, as a flexible linker, a peptide consisting of the GGGGS (SEQ ID NO: 5) sequence, GPGP (SEQ ID NO: 6), and the like. More specifically, as listed in table 3 of Non-Patent Document 19 (Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65 (10): 1357-69). Can be used.

The peptide of the present embodiment and the fusion of the peptide of the present embodiment and the carrier protein (hereinafter, also referred to as “the fusion of the present embodiment”) are not only a chemical synthesis method but also a fusion (the peptide of the present embodiment). − Carrier protein) may be genetically engineered.
First, carrier protein (KLH, etc.) gene information can be easily obtained from a database such as GenBank. Further, as the nucleic acid sequence encoding the peptide of the present embodiment, a person skilled in the art can easily design a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1, and such a nucleic acid sequence. As an example, the nucleic acid sequence represented by SEQ ID NO: 2 can be exemplified.
The fusion of the present embodiment can be prepared, for example, as follows. These nucleic acid sequences (nucleic acid sequences encoding the peptides and carrier proteins of the present embodiment) are arranged so that the peptides and carrier proteins of the present embodiment can be expressed as a fusion linked directly or via a linker. ) Is bound to the encoded DNA (DNA encoding the fusion). The synthesized DNA can be inserted into an appropriate expression vector, the protein can be expressed, and the expressed protein (fusion) can be isolated and purified according to a conventional method.

Expression vectors for expressing the peptide of the present embodiment and the fusion of the present embodiment include, for example, pBR322, pBR325, pUC118, pET and the like (Escherichia coli host), pEGF-C, pEGF-N and the like (animal cell host). , PVL1392, pVL1393, etc. (insect cell host), pG-1, Yep13, pPICZ, etc. (yeast cell host) can be used. These expression vectors have replication origins, selectable markers and promoters suitable for each vector, and optionally include enhancers, transcriptional concentrating sequences (terminators), ribosome binding sites and polyadenylation signals. You may have.

When extracting the expressed peptide or fusion from cultured cells or cells, after culturing, the cells are collected by a known method, suspended in an appropriate buffer solution, and subjected to ultrasonic waves, lysozyme, freeze-thaw, etc. After destroying the cells or cells, a soluble extract is obtained by centrifugation or filtration. From the obtained extract, the desired peptide or fusion can be obtained by appropriately combining known separation / purification methods. Known separation and purification methods include methods that utilize solubility such as salting out and solvent precipitation, dialysis methods, ultrafiltration methods, gel filtration methods, and methods that mainly utilize differences in molecular weight such as SDS-PAGE. A method that utilizes the difference in charge such as ion exchange chromatography, or a method that utilizes specific affinity such as affinity chromatography (for example, when a protein is expressed together with a GST tag, a resin in which glutathione is bound to a carrier is used. , Ni-NTA resin or Co-based resin can be used when protein is expressed with His tag, and anti-HA antibody binding column can be used when protein is expressed with HA tag), reverse. A method utilizing the difference in hydrophobicity such as phase high-speed liquid chromatography, a method utilizing the difference in isoelectric focusing such as isoelectric focusing, and the like are used.

The first embodiment also includes a vaccine for the prevention or treatment of AGEs-RAGE related diseases, which comprises a vector capable of expressing the peptide of the present embodiment or the fusion of the present embodiment.
As the expression vector used here, in addition to the above-mentioned vectors, viral vectors such as adenovirus, adeno-associated virus, lentivirus, and retrovirus can also be preferably used. An expression vector as a vaccine is a promoter (eg, SV40) capable of functioning in vivo, such as an element necessary for expressing the peptide of the present embodiment or a fusion of the peptide of the present embodiment and a carrier protein. It may contain a terminator region, and if necessary, a selection marker gene, etc., in addition to an initial promoter of origin, a viral promoter such as cytomegalovirus LTR, a mammalian constituent protein gene promoter such as β-actin gene promoter, etc.).

The vaccine of this embodiment includes one or more types of adjuvants, Freund's incomplete and complete adjuvants, aluminum salts (eg aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum chloride), oil-in-water emulsions (eg AS03). , MF59®, RibiTM, Provax ^TM ), monophosphoryl lipid A (MPL), potassium myoban, saponin (eg, QS2, QS21, ISCOM (immunostimulatory complex))-containing adjuvants (eg, AS01 (QS2, MPL and) (Including liposomes), AS02 (including squalene, MPL and QS21), AS04 (including MPL and aluminum hydroxide), AS15 (including MPL, QS21, CpG, liposomes), RC-529 (MPL analog), E6020 (including liposomes) Liposomes (liposome A analog)), toxins (eg, mutant CT (non-toxic mutant cholera toxin), mutant LT (non-toxic mutant heat-resistant Escherichia coli toxin), pertussis toxin), flaggerin, adjuvant consisting of nucleic acid components (CpG oligodeoxynucleotide, cyclic di -GMP, cyclic di-AMP, poly (I: C), Ampligen), imidazoquinolin (eg imikimod or R-848), muramildipeptide, trehalose dibechenic acid (eg CAF01), SAF, Ribi, Liposome , Biodegradable microsphere, cytokines (eg, interferon gamma, interleukin 1, interleukin 2, interleukin 12), cyclodextrin, biopolymers (Advax, Inulin polymer, Hemozoin, liposome polymer), chitosan, novasome or nonionic block copolymers Alternatively, DEAE dextran, mineral oil, vegetable oil, etc. can be included. It may also contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier needs to be a compound that does not adversely affect the health of the vaccinated animal. A pharmaceutically acceptable carrier is, for example, sterile water or buffer.
The vaccine preparation of the present embodiment can be administered by a usual active immunization method, and may be a systemic vaccine administered by injection or a mucosal-induced vaccine administered by oral or nasal administration without injection. Good.

When formulating the vaccine of the present embodiment, a known pharmaceutically acceptable stabilizer, preservative, antioxidant, etc. may be formulated together with the vaccine antigen. Examples of the stabilizer include gelatin, dextran, sorbitol and the like. Examples of the preservative include thimerosal, β-propiolactone and the like. Examples of the antioxidant include α-tocopherol and the like.

The dose of the vaccine of the present embodiment can be appropriately determined depending on the age, body weight, etc. of the administration target, but is a pharmaceutically effective amount of the vaccine antigen. A pharmaceutically effective amount refers to the amount of antigen required to induce an immune response against the vaccine antigen. For example, a single vaccine antigen dose of several μg to several tens of mg may be administered once to several times a day, and a total of several times, for example, 1 to 5 times may be administered at intervals of 1 to several weeks.

The second embodiment is the isolated peptide of (a) or (b) below (ie, the isolated peptide of the present embodiment).
One or several amino acids have been substituted, deleted, inserted and / or added in (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or (b) the amino acid sequence represented by SEQ ID NO: 1. A peptide consisting of an amino acid sequence and having the same immunogenicity as the peptide of (a) above. The second embodiment also includes a fusion of the peptide of the present embodiment and a carrier protein. Further, the second embodiment also includes a nucleic acid encoding the peptide of the present embodiment or a fusion of the peptide of the present embodiment and a carrier protein.
The description of the peptide of this embodiment and the carrier protein of this embodiment, which is described in the first embodiment, also applies to the second embodiment.

A third embodiment is a method for preventing and / or treating an AGEs-RAGE system-related disease, which comprises administering the vaccine (or vaccine preparation) of the present embodiment to a patient.
Here, "treatment" means to prevent or alleviate the progression and exacerbation of the pathological condition in a patient who has already developed an AGEs-RAGE-related disease, thereby preventing the progression and exacerbation of the AGEs-RAGE-related disease. A procedure intended to prevent or alleviate.
In addition, "prevention" means to prevent the onset of AGEs-RAGE-related diseases in advance for patients who may develop AGEs-RAGE-related diseases, thereby preventing the onset of AGEs-RAGE-related diseases in advance. It is the intended treatment.

If this specification is translated into English and contains the singular words "a", "an", and "the", not only the singular, unless the context clearly indicates otherwise. It shall include more than one.
Examples are shown below, but the present examples do not limit the scope of the present invention.

1. 1. Materials and experimental methods 1-1. Animal Male DBA2 / JJcl and BKS.Cg- + Lepr ^db / + ^{Lepr db} / Jcl are purchased from CLEA Japan, Inc. (Tokyo, Japan) and are constantly in a standard 12-12 hour light-dark cycle. It was bred under the conditions of feeding and uninterrupted water supply. All experiments were conducted in accordance with the Keio Animal Experiment Regulations and approved by the Keio Animal Experiment Committee (approval number: 16071-1).

1-2. Preparation of RAGE vaccine
The partial peptide of RAGE (GenPept accession no. NP_031451.2; amino acid 38-44, SEQ ID NO: 1) was synthesized by Eurofins Genomics (Tokyo, Japan) and crosslinker, m-maleimidebenzoyl-N-hydroxysuccinimide ester (Thermo). It was ligated to the KLH protein via Fisher Scientific Inc., Waltham, Massachusetts, USA). To prepare a water-in-oil emulsion for enhancing an antigen-specific immune response, add 20 μg of RAGE-KLH antigen (in 100 μl aqueous solution) to 100 μl of Freund's adjuvant (equal to antigen volume) (Sigma- Aldrich Japan, Tokyo, Japan) was mixed. Complete Freund's adjuvant (CFA) was used for the first immunization, and Incomplete Freund's adjuvant (IFA) was used for the subsequent immunization.

1-3. Immune protocol
4-week-old male DBA2 / JJcl mice were divided into two groups. Each group of mice received 3 RAGE vaccines (20 μg RAGE-KLH antigen and Freund's adjuvant; total 200 μl / mouse) or vehicle (20 μg KLH and Freund's adjuvant; total 200 μl / mouse) every 2 weeks. Subcutaneous injections were performed several times (4, 6 and 8 weeks old).
In addition, 10-week-old male db / db mice were divided into two groups, and the mice in each group were subcutaneously injected with the RAGE vaccine or vehicle three times every two weeks (10, 12, and 14 weeks of age).

1-4. Diabetic kidney disease model
Low-dose streptozotocin (40 mg / kg / day) (Sigma-Aldrich Japan) was given to 10-week-old male DBA / 2J mice immunized with the RAGE vaccine or vehicle at 4, 6, and 8 weeks of age. A 0.1 M sodium citrate buffer (pH 4.5) solution containing was administered intraperitoneally to induce diabetes. DBA / 2J mice treated with streptozotocin were used as a type 1 diabetes model, and db / db mice were used as a type 2 diabetes model.

1-5. Evaluation of antibody titer Serum IgG antibody titer was evaluated by the ELISA method (n = 4 / group). Each well of a 96-microwell plate (Immulon 1B; Thermo Fisher Scientific Inc.) was coated with 1.0 μg of RAGE partial peptide (SEQ ID NO: 1) bound bovine serum albumin (BSA) and incubated overnight at 4 ° C. The plate was then incubated with blocking buffer (phosphate buffer containing Tween 20 and 1 w / w% BSA; PBST) for 1 hour before adding serum samples. Serum was diluted with blocking buffer (2-fold serial dilution), added to each well and incubated for 2 hours at 25 ° C. After washing the plates with PBST, goat anti-mouse IgG antibody (1: 5,000 dilution) (SouthernBiotech, Birmingham, Alabama, USA) was added to each well and the plates were incubated for 1.5 hours at 25 ° C. The antigen-antibody reaction was visualized using the TMB Microwell peroxidase substrate system (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Maryland, USA), and _{the absorbance at 450 nm (OD 450} ) was measured. The endpoint titer was shown as a Reciprocal log 2 with a final dilution showing an _{OD 450} of 0.1 units higher than the negative control.

1-6. Biochemical study
Urine samples from type 1 diabetes model (n = 10 / group) and type 2 diabetes model (n = 6 / group) using metabolic cages (type 1 diabetes model 18 weeks old, type 2 diabetes model 28 weeks old) ) Was collected. Urine albumin was measured by direct competitive ELISA according to the instructions for use (Albuwell M; Exocell Inc., Philadelphia, Pennsylvania, USA). Urine creatinine, serum creatinine and serum urea nitrogen were measured enzymatically.

1-7. Optical microscopic analysis The right kidney was removed, fixed with 4 w / w% paraformaldehyde, and then embedded in a paraffin block (type 1 diabetes model n = 8 / group, type 2 diabetes model n = 4 / group). Paraffin sections were stained with periodic acid-Schiff (PAS). The cross-sectional area of at least 30 contiguous glomeruli of any choice was measured to determine the glomerular volume of each mouse. The glomerular volume [V (G)] is
In the same glomerulus, the mesangial substrate region was calculated as the PAS positive staining region, and the ratio of the total glomerular region to the cross section was shown as the mesangial substrate / glomerular area ratio.

1-8. Electron Microscopic Analysis The left kidney was fixed overnight in 0.1 M phosphate buffer (pH 7.4) with 2% paraformaldehyde and 2 w / w% glutaraldehyde at 4 ° C. Then, it was fixed in 0.1 M phosphate buffer with 2 w / w% osmium tetroxide at 4 ° C for 2 hours (type 1 diabetes model n = 4 / group). Kidney samples were dehydrated with a stepwise alcohol solution, impregnated with propylene oxide, embedded in freshly prepared 100% resin and polymerized at 60 ° C. for 48 hours. The polymerized resin was cut into ultrathin sections at 70 nm with a diamond knife using an ultramicrotome, and then the sections were mounted on a copper grid. Sections were stained with 2 w / w% uranyl acetate at room temperature for 15 minutes and secondarily stained with Lead stain solution (Sigma-Aldrich Japan) at room temperature for 3 minutes. The grid was observed using a scanning electron microscope (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) under the condition of an acceleration voltage of 100 kV. The digital image (3296 x 2472 pixels) was taken with a CCD camera (EM-14830RUBY2; JOEL Ltd.).
The thickness of the glomerular basement membrane (GBM) was determined using the orthogonal intercept method (Steenhard et al., Am J Pathol. 177: 84-96 2010). 32-40 digital images of 16,800 x magnification were stored for each animal (1 image per snare, 8-10 snares per glomerulus, 4 glomeruli per animal). A grid mask with 16 intercepts was applied to each image using Photoshop CS4 (Adobe Systems Inc., San Jose, California, USA). GBM thickness was measured in each section from the basement membrane endothelial cell membrane to the basement membrane podocyte cell membrane.

2. Result 2-1. Induction of RAGE Partial Peptide (SEQ ID NO: 1) -Specific Antibody Male DBA2J mice were injected subcutaneously with RAGE (SEQ ID NO: 1) -KLH fusion 20 μg + each adjuvant (FCA / FIA, Alum or polyIC) at 4, 6 and 8 weeks of age. After administration, blood was collected at 10 weeks of age, and the serum IgG antibody titer against the antigen was measured (Fig. 1). The antibody titer was the highest when Freund's adjuvant was used as the adjuvant, and the antibody titer was also high when polyIC was used.
Next, the change over time in the IgG antibody titer against the RAGE partial peptide was examined. As a result, the antibody titer was below the measurement sensitivity at the end of the first immunization, but the antibody titer increased after the second inoculation and showed a higher antibody titer after the end of the third administration. After that, the antibody titer was maintained at a relatively high value without diminishing (Fig. 2).
From this result, it is considered that the vaccine of the present embodiment has an effect of continuously inducing an antibody in vivo.

Analysis using 2-2.1 type diabetes model mice 2-2-1. Changes in blood glucose levels in type 1 diabetes model mice after administration of RAGE peptide vaccine (RAGE-KLH).
Male DBA2J mice were injected subcutaneously with RAGE-KLH 20 μg + Freund's adjuvant at 4, 6 and 8 weeks of age, and streptozotocin was administered at 10 weeks of age to induce diabetes (type 1 diabetes model) (KLH 20 μg + Freund as vehicle). Administer adjuvant). Hyperglycemia was observed 2 weeks after streptozotocin administration (Fig. 3, left figure), and even higher blood glucose level was observed 8 weeks after administration (Fig. 3, right figure). That is, it was found that there was no difference in blood glucose level between the vaccinated group and the vehicle inoculated group (according to unpaired t-test).

2-2-2. Suppressive effect of albuminuria by RAGE peptide vaccine in type 1 diabetes model mice.
2-2-1 above. Similarly, diabetes was induced in male DBA2J mice. Urinary albumin excretion 8 weeks after streptozotocin administration was significantly lower in the vaccinated group (850.6 ± 208.6 in the vehicle vaccinated group, 333.6 ± 104.7 in the vaccinated group) (Fig. 4).

Inhibitory effect of RAGE peptide vaccine on glomerular swelling and mesangial matrix expansion in type 2-2-3.1 diabetes model mice.
2-2-1 above. Similarly, diabetes was induced in male DBA2J mice. Observation of renal tissue 12 weeks after streptozotocin administration revealed suppression of glomerular swelling (glomerular volume) in the vaccinated group (vehicle vaccinated group 5.28 ± 0.52, vaccinated group 3.18 ± 0.17), and mesangium substrate expansion (mesangium substrate). Suppression of / glomerular area) (vehicle inoculation group 26.3 ± 1.6, vaccination group 18.8 ± 0.8) was observed (Fig. 5).

2-2-4.1 Inhibitory effect of glomerular basement membrane thickening by RAGE peptide vaccine in type 1 diabetes model mice.
2-2-1 above. Similarly, diabetes was induced in male DBA2J mice. When the kidneys 12 weeks after the administration of streptozotocin were observed with an electron microscope, it was found that the thickening of the glomerular basement membrane was suppressed in the vaccinated group (vehicle vaccinated group 215.3 ± 1.9, vaccinated group 181.3 ± 1.8) (Fig.) 6).

Analysis by type 2-3.2 diabetes model mice 2-3-1.2 Effect of RAGE peptide vaccine on albuminuria in type 2 diabetes model mice.
Male db / db mice (type 2 diabetes model mice) were injected subcutaneously with RAGE-KLH 20 μg + Freund's adjuvant at 10, 12 and 14 weeks of age (KLH 20 μg + Freund's adjuvant was administered as a vehicle). Urinary albumin excretion immediately before immunization (at 10 weeks of age) was similar in both groups (vehicle inoculation group 403.0 ± 55.3, vaccination group 469.5 ± 99.1), but urine in the vaccination group 14 weeks after the final immunization. The increase in middle albumin excretion was significantly suppressed (vehicle inoculation group 737.7 ± 116.4, vaccination group 367.0 ± 88.2) (Fig. 7).

2-3-2.2 Inhibitory effect of RAGE peptide vaccine on glomerular swelling and mesangial matrix expansion in type 2 diabetes model mice.
2-3-1 above. Similarly, the vaccine or vehicle was administered by subcutaneous injection. At 20 weeks (34 weeks of age) after final immunization, mouse kidneys were evaluated by PAS staining. Suppression of glomerular swelling (glomerulus volume) in the vaccinated group (vaccination group 5.67 ± 0.18, vaccination group 3.22 ± 0.23), and suppression of mesangium substrate expansion (mesangium substrate / glomerular area) (vehicle inoculation group 32.7) ± 2.3, vaccination group 23.6 ± 1.0) was observed (Fig. 8).

The above results are obtained when the vaccine of the present embodiment is administered to mice before the onset of diabetes (analysis using streptozotocin-induced diabetic mice) and when it is administered to mice after the onset of diabetes (analysis using db / db mice). ), It is shown that various symptoms associated with diabetic kidney disease are alleviated.
Therefore, the vaccine of the present embodiment is considered to have an effect as a vaccine for the prevention or treatment of diseases related to AGEs-RAGE hyperactivity.

The vaccine of the present embodiment has a preventive and therapeutic effect on the pathological condition of diabetic kidney disease, which is one of AGEs-RAGE related diseases. Therefore, the present invention is expected to be used in the medical field.

Claims (5)

A vaccine for preventing or treating AGEs-RAGE-related diseases, which comprises the following peptides (a) or (b).
One or several amino acids have been substituted, deleted, inserted and / or added in (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or (b) the amino acid sequence represented by SEQ ID NO: 1. A peptide consisting of an amino acid sequence and having the same immunogenicity as the peptide of (a) above. The vaccine according to claim 1, wherein the carrier protein is fused to the peptides (a) and (b). The vaccine according to claim 1 or 2, which comprises a vector capable of expressing the peptide of (a) or (b) or a fusion of these peptides and a carrier protein. The isolated peptide of (a) or (b) below.
One or several amino acids have been substituted, deleted, inserted and / or added in (a) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or (b) the amino acid sequence represented by SEQ ID NO: 1. A peptide consisting of an amino acid sequence and having the same immunogenicity as the peptide of (a) above. A nucleic acid encoding the peptide according to claim 4.