JP2021136920A - Antibody against glycation end product, and use of the same - Google Patents

Abstract

To provide a monoclonal antibody having high selectivity and affinity to AGEs, particularly glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs, and an analytic method utilizing the antibody, and furthermore to provide a diagnostic method, a therapeutic method and a prevention method of a disease using the monoclonal antibody.SOLUTION: There is provide a monoclonal antibody and an analytic method using the antibody, and a diagnostic method, a therapeutic method and a prevention method of a disease.SELECTED DRAWING: Figure 30

Description

The present invention relates to advanced glycation end products (AGEs), particularly monoclonal antibodies or antigen-binding fragments thereof that bind to epitopes of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs.

Advanced glycation end products (AGEs) are a general term for substances produced in vivo through complex reactions such as oxidation, dehydration, and condensation after the amino groups of amino acids, mainly lysine, are non-enzymatically modified by reducing sugars. be. AGEs produced by aging and hyperglycemia are considered to be associated with lifestyle-related diseases such as diabetic complications and arteriosclerosis. In particular, AGEs (advanced glycation end products; Glycer-AGEs) derived from glyceraldehyde, which is a sugar metabolism intermediate, are attracting attention as useful biomarkers in diagnosis and prevention (Non-Patent Documents 1 to 3).

Several structures contained in AGEs have been identified, and one of them, carboxymethyl lysine (N ^ε- (carboxymethyl) lysine, CML), has a transition of the sugar portion of the Amadori compound produced by the reaction of glucose and lysine. It has been identified as a substance that oxidatively cleaves in the presence of a metal and is produced with erythronic acid. From the history of AGEs research, quantification of AGEs has been replaced by quantification of CML whose structure has been clarified. The main pathway for CML production in vitro is believed to be due to oxidative cleavage of Schiff bases or Amadori compounds. It is also known that CML is also produced by an intramolecular cannizzaro reaction using glyoxal (GO) and glycolaldehyde as precursors and autoxidation of glucose. CML in protein may be stable to acid hydrolysis and can be quantified by high performance liquid chromatography (HPLC) method or gas chromatography / mass spectrometry (GC / MS) method (Non-Patent Document 1). .. Several reports have been made on the structures contained in AGEs (Patent Documents 1 to 3).

As a quantification of AGEs by an enzyme-linked immunosorbent assay (EIA) method, a competitive enzyme using an anti-AGEs-BSA antibody using AGEs (AGEs-containing bovine serum albumin, AGEs-BSA) prepared using bovine serum albumin (BSA) as an antigen. Starting with the -linked immunosorbent assay (ELISA) method, these antibodies were later found to be anti-CML antibodies having a CML structure as an epitope (Non-Patent Document 1). On the other hand, antisera are prepared using AGEs prepared by adding glyceraldehyde, glycolaldehyde, methylglyoxal, or glyoxal to rabbit serum albumin (RSA) as an antigen, and the obtained antiserum is purified to be non-CML. It has been reported that a binding fraction was obtained (Non-Patent Document 4). An ELISA assay using the polyclonal antibody obtained as the fraction has been performed, and it is used as a method for quantifying AGEs other than CML (Non-Patent Documents 5 and 6).

For example, studies have been conducted on the correlation between the success or failure of fertility treatment and glyceraldehyde-derived AGEs (Glycer-AGEs), and it has been reported that a high amount of Glycer-AGEs in the blood results in a poor continuous pregnancy rate. (Non-Patent Documents 1, 3, 5 and 6). It has also been reported that AGEs induce endothelial-to-mesenchymal transitions, and that epithelial-mesenchymal transitions are involved in the development of cardiovascular diseases and fibrotic diseases (Non-Patent Document 13). And 14). In such studies, ELISA assays using anti-glyceraldehyde-derived AGEs antibodies among the above-mentioned polyclonal antibodies are utilized (Non-Patent Documents 5 and 6). Several reports have been made on antibodies having binding activity to AGEs (Patent Documents 4 to 6).

Many reports have been made on the specific chemical structures of various AGEs (for example, Non-Patent Documents 8 to 12 and 15). In addition, there has been a report on the production of monoclonal antibodies using AGEs derived from glyceraldehyde as antigens (Non-Patent Document 7).

Japanese Unexamined Patent Publication No. 2001-316389; Japanese Patent Application Laid-Open No. 2003-30961; Japanese Unexamined Patent Publication No. 2004-250404; Japanese Unexamined Patent Publication No. 2006-31621; JP-A-2017-043595; JP-A-2019-041668;

Kanazawa Medical University Magazine, 37 (4): 141-161, 2012; Kanazawa Medical University Magazine, 40 (2/3): 95-103, 2015; Diagnostics 6 (2), 23, 2016; doi: 10.3390; Molecular Medicine, 6 (2): 114-125, 2000; Human Reproduction, 26 (3), 604-610, 2011; Journal of Japan Society for Mibyou Systems, 21 (1): 93-96, 2015; Immunology Letters 167 (2), 141-146, 2015; Biosci. Biotechnol. Biochem. 67 (4), 930-932, 2003; J. Agric. Food Chem. 47 (2), 379-390, 1999; J. Agric. Food Chem. 25 (6), 1282-1287, 1977; Agric. Biol. Chem.49 (11), 3131-3137; Biochem. J. 369 (3), 705-719, 2003; Biomed. Res. Int. 684242,2015; Int. J. Mol. Sci. 18 (10), 2017. Free Radic. Biol. Med. 29 (6), 557-567, 2000.

An object of the present invention is to provide a monoclonal antibody having high selectivity and affinity for AGEs, particularly AGEs derived from glyceraldehyde or AGEs derived from glycolaldehyde, and an analysis method using the same. A further object of the present invention is to provide a monoclonal antibody having a chemical structure contained in AGEs that damages a living body or causes a disease as an epitope, and an analysis method using the same. Furthermore, an object of the present invention is to provide a method for diagnosing, treating and preventing a disease using the monoclonal antibody, particularly a method for diagnosing, treating and preventing an infertility or an eye disease.

As a result of research, the present inventors have found that a specific monoclonal antibody has good selectivity and high binding property to an antigen and can be used for analytical methods, diagnostic methods, therapeutic methods and preventive methods. , The present invention has been completed.

The present invention provides the following inventions.

[1] (1) Amino acid sequences of complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3) of heavy chain variable regions or complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3) of light chain variable regions. but,
(1-3H)
(A) VH CDR1: The amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence substantially the same as that;
(B) VH CDR2: Amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence substantially identical thereto; and (c) VH CDR3: Amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence substantially identical thereto. ; Or (1-3L)
(D) VL CDR1: The amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence substantially the same as that;
(E) VL CDR2: Amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence substantially the same as that; and (f) VL CDR3: Amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence substantially the same thereof. ;
A monoclonal antibody or antigen-binding fragment thereof, which comprises.

[2] (1) Amino acid sequences of the complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3) of the heavy chain variable region and the complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3) of the light chain variable region. but,
(A) VH CDR1: The amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence substantially the same as that;
(B) VH CDR2: Amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence substantially the same as that;
(C) VH CDR3: Amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence substantially the same as that;
(D) VL CDR1: The amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence substantially the same as that;
(E) VL CDR2: Amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence substantially the same as that; and (f) VL CDR3: Amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence substantially the same thereof. ;
The monoclonal antibody or antigen-binding fragment thereof according to [1], which comprises.

[3] (1) The amino acid sequences of the heavy chain variable region and the light chain variable region are
The amino acid sequence of SEQ ID NO: 26 or substantially the same amino acid sequence, and the amino acid sequence of SEQ ID NO: 32 or substantially the same amino acid sequence;
The amino acid sequence of SEQ ID NO: 27 or substantially the same amino acid sequence and / or the amino acid sequence of SEQ ID NO: 33 or substantially the same amino acid sequence;
The amino acid sequence of SEQ ID NO: 28 or substantially the same amino acid sequence and / or the amino acid sequence of SEQ ID NO: 34 or substantially the same amino acid sequence;
The monoclonal antibody or antigen-binding fragment thereof according to [1] or [2], which comprises.

[4] The monoclonal antibody according to any one of [1] to [5] or an antigen-binding fragment thereof, which binds to an epitope contained in glyceraldehyde-derived AGEs.

［式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択され、
Ｘ^１およびＸ^２は、−Ｏ−、または−ＮＨ−を表し；
Ｙ^１およびＹ^２は、水素原子、保護基、または基：

を表し；
Ｒ^５およびＲ^６は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択される］
の化合物、そのカチオンラジカル、またはそのジカチオンと結合する、［１］〜［５］のいずれかに記載のモノクローナル抗体またはその抗原結合断片。 [5] Equation (I) or (II):

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues]
The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [5], which binds to the compound, its cation radical, or its dication.

[6] The full-length antibody, Fab, Fab', F (ab') ₂ , Fv, scFv, dsFv, diabody, or sc (Fv) ₂ , according to any one of [1] to [6]. Monoclonal antibody or antigen-binding fragment thereof.

[7] The monoclonal antibody or an antigen-binding fragment thereof according to any one of [1] to [6], which is a mouse antibody, a humanized antibody, a human antibody, a chimeric antibody, or an antigen-binding fragment thereof.

[8] The monoclonal antibody according to any one of [1] to [7], which binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs with a dissociation constant (K _d ^{value) of 1 × 10 -5 M or less.} Or its antigen-binding fragment.

[9] A pharmaceutical composition containing the monoclonal antibody according to any one of [1] to [8] or an antigen-binding fragment thereof.

[10] Obesity, diabetes, diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic myocardium, diabetic vascular complications, diabetic nephropathy, diabetic kidney disease, diabetic foot lesions, diabetic ketoacidosis, periodontal disease, addition Age-yellow spot degeneration, pulmonary fibrosis, idiopathic pulmonary fibrosis, peribronchial fibrosis, interstitial lung disease, lung cancer, cancer fibrosis, chronic obstructive pulmonary disease, acute lower extremity arterial embolism, peripheral arteries Diseases, peripheral airway disease, pulmonary emphysema, nephritis, glomerulosclerosis, glomerulonephritis, mesangial proliferative glomerulonephritis, diabetic nephropathy, renal systemic fibrosis, chronic kidney disease, idiopathic retroperitoneal fibrosis, renal disease , Strong skin disease, renal interstitial fibrosis, female infertility, polycystic ovary syndrome, ovarian insufficiency, early ovarian insufficiency, ovarian cancer, breast cancer, uterine body cancer, prostate cancer, male infertility, liver Diseases, liver cirrhosis, non-alcoholic steatosis, liver cancer, atherosclerotic cerebral infarction, atherosclerosis, internal carotid artery stenosis, aortic valve stenosis, aortic valve insufficiency, cardiovascular disease, angina , Congestive heart failure, acute heart failure, chronic heart failure, ischemic heart disease, dilated cardiomyopathy, cardiac sarcoidosis, hypertension, pulmonary arterial pulmonary hypertension, pulmonary heart, myocarditis, vascular stenosis heart fibrosis, post-myocardial infarction heart fiber Disease, left ventricular hypertrophy after myocardial infarction, rheumatoid arthritis, lifestyle disease, dyslipidemia, Alzheimer's disease, vascular dementia, cerebral infarction, brain tumor, cerebrovascular disorder, fibrosis, endocrine disease, osteoporosis, tongue Oral cancer, pharyngeal cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, retinal pigment degeneration, diabetic luteal edema, Labor congenital melanosis, Stargart's disease, Asher syndrome, colloideremia, Rod pyramidal dystrophy, pyramidal dystrophy, progressive retinal atrophy, luteal dystrophy, choroidal sclerosis, total choroidal atrophy, cystic edema, vegetitis, retinal detachment, luteal foramen, luteal capillary dilatation The medicament according to [9], which is used for diagnosing, treating, or preventing a disease selected from glaucoma, optic neuropathy, ischemic retinal disease, premature infant retinosis, retinal vascular occlusion, and retinal aneurysm. Composition.

[11] Diabetes mellitus, impaired glucose tolerance, retinopathy, nephropathy, complications associated with diabetes mellitus, peripheral neuropathy, lower limb necrosis, arteriosclerosis, thrombosis, non-alcoholic fatty liver disease, non-alcoholic steatosis, cancer The medicament according to [9], which is used for diagnosing, treating, or preventing a disease selected from neurodegenerative diseases including infertility, polycystic ovary syndrome, ovarian dysfunction, central neuropathy, and Alzheimer's disease. Composition.

[12] The pharmaceutical composition according to [11], wherein the disease is cancer selected from melanoma, lung cancer, and liver cancer.

[13] For use in diagnosis, treatment, or prevention of diseases selected from diabetic nephropathy, nephritis, glomerulonephritis, glomerulonephritis, renal systemic fibrosis, chronic kidney disease, and renal interstitial fibrosis. The pharmaceutical composition according to [9].
[14] The pharmaceutical composition according to [9] for use in the diagnosis, treatment, or prevention of eye diseases.

[15] Eye diseases include diabetic retinopathy, diabetic cataract, retinal pigment degeneration, diabetic macular edema, Labor congenital melanosis, Stargart's disease, Asher syndrome, colloideremia, rod pyramidal dystrophy, pyramidal dystrophy, progressive retinal atrophy. , Age-related macular degeneration, macular dystrophy, choriosclerosis, total choroidal atrophy, cystic macular edema, vaginitis, retinal detachment, macular foramen, macular capillary dilatation, glaucoma, optic neuropathy, ischemic The pharmaceutical composition according to [14], which is selected from retinal diseases, premature infant retina, retinal vascular occlusion, and retinal macula.

[16] A nucleic acid encoding the monoclonal antibody according to any one of [1] to [8] or an antigen-binding fragment thereof.
[17] An expression vector containing the nucleic acid according to [16].
[18] A host cell containing the expression vector of [17].

[A1] 1) The amino acid sequences of the complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3) of the heavy chain variable region and the complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3) of the light chain variable region are ,
(A) The amino acid sequence shown in VH CDR1: SEQ ID NO: 1 or an amino acid sequence substantially the same as that;
(B) VH CDR2: Amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence substantially the same as that;
(C) VH CDR3: Amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence substantially the same as that;
(D) VL CDR1: The amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence substantially the same as that;
(E) VL CDR2: Amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence substantially the same as that;
(F) VL CDR3: A monoclonal antibody or an antigen-binding fragment thereof, which comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence substantially identical thereto.
Or 2) An antibody or an antigen-binding fragment thereof that cross-competes with the antibody or an antigen-binding fragment thereof with respect to binding to an epitope of AGEs derived from glyceraldehyde or AGEs derived from glycolaldehyde.
A monoclonal antibody or an antigen-binding fragment thereof that binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs.

［式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択され、
Ｘ^１およびＸ^２は、−Ｏ−、または−ＮＨ−を表し；
Ｙ^１およびＹ^２は、水素原子、保護基、または基：

を表し；
Ｒ^５およびＲ^６は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択される］
の化合物、そのカチオンラジカル、またはそのジカチオンと結合するモノクローナル抗体またはその抗原結合断片。 [A2] Formula (I) or (II):

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues]
Compound, its cation radical, or a monoclonal antibody or its antigen-binding fragment that binds to its dication.

[A3] 1) The amino acid sequences of the heavy chain variable region and the light chain variable region are
SEQ ID NO: 4 or substantially the same amino acid sequence, and SEQ ID NO: 8 or substantially the same amino acid sequence; or 2) Glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs epitopes The monoclonal antibody or antigen-binding fragment thereof according to [A1], which is an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof with respect to binding to.

[A4] The monoclonal antibody or antigen-binding fragment thereof according to any one of [A1] to [A3], wherein the epitope is an epitope of AGEs derived from glyceraldehyde.
[A5] The full-length antibody, Fab, Fab', F (ab') ₂ , Fv, scFv, dsFv, diabody, or sc (Fv) ₂ , according to any one of [A1] to [A4]. Monoclonal antibody or antigen-binding fragment thereof.

[A6] The monoclonal antibody or the antigen-binding fragment thereof according to any one of [A1] to [A5], which is a mouse antibody, a humanized antibody, a human antibody, a chimeric antibody, or an antigen-binding fragment thereof.
[A7] The monoclonal antibody according to any one of [A1] to [A6], which binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs with a dissociation constant (K _d ^{value) of 1 × 10-5 M or less.} Or its antigen-binding fragment.

[A8] A pharmaceutical composition containing the monoclonal antibody according to any one of [A1] to [A7] or an antigen-binding fragment thereof.
[A9] Obesity, diabetes, diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic myocardium, diabetic vascular complications, diabetic nephropathy, diabetic kidney disease, diabetic foot lesions, diabetic ketoacidosis, periodontal disease, addition Age-yellow spot degeneration, pulmonary fibrosis, idiopathic pulmonary fibrosis, peribronchial fibrosis, interstitial lung disease, lung cancer, cancer fibrosis, chronic obstructive pulmonary disease, acute lower extremity arterial embolism, peripheral arteries Diseases, peripheral airway disease, pulmonary emphysema, nephritis, glomerulosclerosis, glomerulonephritis, mesangial proliferative glomerulonephritis, diabetic nephropathy, renal systemic fibrosis, chronic kidney disease, idiopathic retroperitoneal fibrosis, renal disease , Strong skin disease, renal interstitial fibrosis, female infertility, polycystic ovary syndrome, ovarian dysfunction, early ovarian dysfunction, ovarian cancer, breast cancer, uterine body cancer, prostate cancer, male infertility, liver Diseases, liver cirrhosis, non-alcoholic steatosis, liver cancer, atherosclerotic cerebral infarction, atherosclerosis, internal carotid artery stenosis, aortic valve stenosis, aortic valve insufficiency, cardiovascular disease, angina , Congestive heart failure, acute heart failure, chronic heart failure, ischemic heart disease, dilated cardiomyopathy, cardiac sarcoidosis, hypertension, pulmonary arterial pulmonary hypertension, pulmonary heart, myocarditis, vascular stenosis heart fibrosis, post-myocardial infarction heart fiber Disease, left ventricular hypertrophy after myocardial infarction, rheumatoid arthritis, lifestyle disease, dyslipidemia, Alzheimer's disease, vascular dementia, cerebral infarction, brain tumor, cerebrovascular disorder, fibrosis, endocrine disease, osteoporosis, tongue Described in [A8] for use in diagnosing, treating, or preventing a disease selected from oral cancer, pharyngeal cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, and pancreatic cancer. Pharmaceutical composition.

[A10] Diabetes mellitus, impaired glucose tolerance, retinopathy, nephropathy, diabetic complications, peripheral neuropathy, lower limb necrosis, arteriosclerosis, thrombosis, non-alcoholic fatty liver disease, non-alcoholic steatosis, cancer The medicament according to [A8] for use in diagnosing, treating, or preventing a disease selected from neurodegenerative diseases including infertility, polycystic ovary syndrome, ovarian dysfunction, central neuropathy, and Alzheimer's disease. Composition.

[A11] The pharmaceutical composition according to [A8], wherein the disease is cancer selected from melanoma, lung cancer, and liver cancer.
[A12] A nucleic acid encoding the monoclonal antibody according to any one of [A1] to [A7] or an antigen-binding fragment thereof.
[A13] An expression vector containing the nucleic acid according to [A12].
[A14] A host cell containing the expression vector of [A13].

［式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択され、
Ｘ^１およびＸ^２は、−Ｏ−、または−ＮＨ−を表し；
Ｙ^１およびＹ^２は、水素原子、保護基、または基：

を表し；
Ｒ^５およびＲ^６は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択される］
の化合物、そのカチオンラジカル、またはそのジカチオン、またはその塩。 [A15] Formula (I) or (II):

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues]
Compound, its cation radical, or its dication, or its salt.

［式中、Ｒ^１、Ｒ^３、およびＲ^６は、保護基である］
で表される化合物を含む画分を得ること
３）当該画分を動物への免疫を行い、当該画分を抗原とする抗体を得ること
を含む、抗体の製造方法。 [A16] 1) A reaction mixture is obtained by reacting lysine and glyceraldehyde in which the amino group at the α-position is protected;
2) Fractionate the reaction mixture and formula (Ia), (Ib), (IIa) ”, or (IIb) :.

[In the formula, R ¹ , R ³ , and R ⁶ are protecting groups]
3) A method for producing an antibody, which comprises immunizing an animal with the fraction and obtaining an antibody using the fraction as an antigen.

［式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択され、
Ｑ^１は、水素原子、または基−ＣＨ_２−Ｘ^１−Ｙ^１を表し；
Ｑ^２は、水素原子、または基−ＣＨ_２−Ｘ^２−Ｙ^２を表し；
Ｘ^１およびＸ^２は、−Ｏ−、または−ＮＨ−を表し；
Ｙ^１およびＹ^２は、水素原子、保護基、または基：

を表し；
Ｒ^５およびＲ^６は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択される］
の化合物、そのカチオンラジカル、またはそのジカチオンと結合するモノクローナル抗体またはその抗原結合断片。 [B1] Equation (XI) or (XII):

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
Q ¹ represents a hydrogen atom or group -CH _2- X ^1- Y ¹ ;
Q ² represents a hydrogen atom or group -CH _2- X ^2- Y ² ;
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues]
Compound, its cation radical, or a monoclonal antibody or its antigen-binding fragment that binds to its dication.

［式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択され、
Ｘ^１およびＸ^２は、−Ｏ−、または−ＮＨ−を表し；
Ｙ^１およびＹ^２は、水素原子、保護基、または基：

を表し；
Ｒ^５およびＲ^６は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択される］
の化合物、そのカチオンラジカル、またはそのジカチオンと結合する、［Ｂ１］に記載のモノクローナル抗体またはその抗原結合断片。 [B2] Equation (I) or (II):

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues]
The monoclonal antibody or antigen-binding fragment thereof according to [B1], which binds to the compound, its cation radical, or its dication.

[B3] (1) Amino acid sequences of complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3) of heavy chain variable regions or complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3) of light chain variable regions. but,
(1-1H)
(A) The amino acid sequence shown in VH CDR1: SEQ ID NO: 1 or an amino acid sequence substantially the same as that;
(B) VH CDR2: Amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence substantially the same as that; and (c) VH CDR3: Amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence substantially the same as that. ;
(1-1L)
(D) VL CDR1: The amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence substantially the same as that;
(E) VL CDR2: Amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence substantially the same as that; and (f) VL CDR3: Amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence substantially the same as the amino acid sequence. ;
(1-2H)
(G) VH CDR1: The amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence substantially the same as that;
(H) VH CDR2: Amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence substantially identical thereto; and (i) VH CDR3: Amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence substantially identical thereto. ; Or (1-2L)
(J) VL CDR1: The amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence substantially the same as that;
(K) VL CDR2: Amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence substantially the same as that;
(L) VL CDR3: Amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence substantially the same as that;
Monoclonal antibody or antigen-binding fragment thereof, including;
Or (2) a monoclonal antibody or an antigen-binding fragment thereof that cross-competes with the monoclonal antibody of (1) above or an antigen-binding fragment thereof with respect to binding to an epitope of AGEs derived from glyceraldehyde or AGEs derived from glycolaldehyde.
A monoclonal antibody or an antigen-binding fragment thereof that binds to an epitope contained in glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs.

[B4] (1) Amino acid sequences of the complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3) of the heavy chain variable region and the complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3) of the light chain variable region. but,
(1-1)
(A) The amino acid sequence shown in VH CDR1: SEQ ID NO: 1 or an amino acid sequence substantially the same as that;
(B) VH CDR2: Amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence substantially the same as that;
(C) VH CDR3: Amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence substantially the same as that;
(D) VL CDR1: The amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence substantially the same as that;
(E) VL CDR2: Amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence substantially the same as that; and (f) VL CDR3: Amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence substantially the same as the amino acid sequence. Or (1-2)
(G) VH CDR1: The amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence substantially the same as that;
(H) VH CDR2: Amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence substantially the same as that;
(I) VH CDR3: Amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence substantially the same as that;
(J) VL CDR1: The amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence substantially the same as that;
(K) VL CDR2: Amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence substantially the same as that;
(L) VL CDR3: Amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence substantially the same as that;
Monoclonal antibody or antigen-binding fragment thereof, including;
Or (2) a monoclonal antibody or an antigen-binding fragment thereof that cross-competes with the monoclonal antibody of (1) above or an antigen-binding fragment thereof with respect to binding to an epitope of AGEs derived from glyceraldehyde or AGEs derived from glycolaldehyde.
The monoclonal antibody or antigen-binding fragment thereof according to any one of [B1] to [B3], which binds to an epitope contained in glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs.

[B5] (1) The amino acid sequences of the heavy chain variable region and the light chain variable region are
The amino acid sequence of SEQ ID NO: 4 or substantially the same amino acid sequence and / or the amino acid sequence of SEQ ID NO: 8 or substantially the same amino acid sequence; or the amino acid sequence of SEQ ID NO: 18 or substantially the same thereof. Identical amino acid sequence and / or amino acid sequence of SEQ ID NO: 22 or substantially identical amino acid sequence;
Or (2) an antibody or an antigen-binding fragment thereof that cross-competes with the antibody described in (1) above or an antigen-binding fragment thereof with respect to binding to an epitope of AGEs derived from glyceraldehyde or AGEs derived from glycolaldehyde.
The monoclonal antibody or antigen-binding fragment thereof according to any one of [B1] to [B4], which binds to an epitope contained in glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs.

[B6] The monoclonal antibody according to any one of [B1] to [B5] or an antigen-binding fragment thereof, which binds to an epitope contained in glyceraldehyde-derived AGEs.

[B7] The full-length antibody, Fab, Fab', F (ab') ₂ , Fv, scFv, dsFv, diabody, or sc (Fv) ₂ , according to any one of [B1] to [B6]. Monoclonal antibody or antigen-binding fragment thereof.

[B8] The monoclonal antibody or the antigen-binding fragment thereof according to any one of [B1] to [B7], which is a mouse antibody, a humanized antibody, a human antibody, a chimeric antibody, or an antigen-binding fragment thereof.

[B9] The monoclonal antibody according to any one of [B1] to [B8], which binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs with a dissociation constant (K _d ^{value) of 1 × 10 -5 M or less.} Or its antigen-binding fragment.

[B10] A pharmaceutical composition comprising the monoclonal antibody according to any one of [B1] to [B9] or an antigen-binding fragment thereof.
[B11] Obesity, diabetes, diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic myocardium, diabetic vascular complications, diabetic nephropathy, diabetic kidney disease, diabetic foot lesions, diabetic ketoacidosis, periodontal disease, addition Age-yellow spot degeneration, pulmonary fibrosis, idiopathic pulmonary fibrosis, peribronchial fibrosis, interstitial lung disease, lung cancer, cancer fibrosis, chronic obstructive pulmonary disease, acute lower extremity arterial embolism, peripheral arteries Diseases, peripheral airway disease, pulmonary emphysema, nephritis, glomerulosclerosis, glomerulonephritis, mesangial proliferative glomerulonephritis, diabetic nephropathy, renal systemic fibrosis, chronic kidney disease, idiopathic retroperitoneal fibrosis, renal disease , Strong skin disease, renal interstitial fibrosis, female infertility, polycystic ovary syndrome, ovarian dysfunction, early ovarian dysfunction, ovarian cancer, breast cancer, uterine body cancer, prostate cancer, male infertility, liver Diseases, liver cirrhosis, non-alcoholic steatosis, liver cancer, atherosclerotic cerebral infarction, atherosclerosis, internal carotid artery stenosis, aortic valve stenosis, aortic valve insufficiency, cardiovascular disease, angina , Congestive heart failure, acute heart failure, chronic heart failure, ischemic heart disease, dilated cardiomyopathy, cardiac sarcoidosis, hypertension, pulmonary arterial pulmonary hypertension, pulmonary heart, myocarditis, vascular stenosis heart fibrosis, post-myocardial infarction heart fiber Disease, left ventricular hypertrophy after myocardial infarction, rheumatoid arthritis, lifestyle disease, dyslipidemia, Alzheimer's disease, vascular dementia, cerebral infarction, brain tumor, cerebrovascular disorder, fibrosis, endocrine disease, osteoporosis, tongue Oral cancer, pharyngeal cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, retinal pigment degeneration, Labor congenital melanosis, Stargart's disease, Asher syndrome, colloideremia, rod pyramid Dystrophy, pyramidal dystrophy, progressive retinal atrophy, luteal dystrophy, choriosclerosis, total choroidal atrophy, cystic erythema edema, vasculitis, retinal detachment, luteal foramen, luteal capillary dilatation, glaucoma, optic nerve The pharmaceutical composition according to [B10] for use in the diagnosis, treatment, or prevention of diseases selected from diseases, ischemic retinal diseases, premature infant retinosis, retinal vascular occlusion, and retinal aneurysms.

[B12] Diabetes mellitus, impaired glucose tolerance, retinopathy, nephropathy, diabetic complications, peripheral neuropathy, lower limb necrosis, arteriosclerosis, thrombosis, non-alcoholic fatty liver disease, non-alcoholic steatosis, cancer The medicament according to [B10] for use in diagnosing, treating, or preventing a disease selected from neurodegenerative diseases including infertility, polycystic ovary syndrome, ovarian dysfunction, central neuropathy, and Alzheimer's disease. Composition.

[B13] The pharmaceutical composition according to [B12], wherein the disease is cancer selected from melanoma, lung cancer, and liver cancer.
[B14] The pharmaceutical composition according to [B10] for use in the diagnosis, treatment, or prevention of eye diseases.

[B15] Eye diseases include diabetic retinopathy, diabetic cataract, retinal pigment degeneration, diabetic macular edema, Laver congenital melanosis, Stargart's disease, Asher syndrome, colloideremia, rod pyramidal dystrophy, pyramidal dystrophy, and progressive retinal atrophy. , Age-related macular degeneration, macular dystrophy, choriosclerosis, total choroidal atrophy, cystic macular edema, vaginitis, retinal detachment, macular foramen, macular capillary dilatation, glaucoma, optic neuropathy, ischemic The pharmaceutical composition according to [B14], which is selected from retinal diseases, premature infant retina, retinal vascular occlusion, and retinal macula.

[B16] A nucleic acid encoding the monoclonal antibody according to any one of [B1] to [B9] or an antigen-binding fragment thereof.
[B17] An expression vector containing the nucleic acid according to [B16].
A host cell containing the expression vector of [B18] and [B17].
[B19] Formula (I) or (II):

［式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、それぞれ独立に、水素原子、保護基、および
アミノ酸残基数１〜１０００のペプチド基から選択され、
Ｘ^１およびＸ^２は、−Ｏ−、または−ＮＨ−を表し；
Ｙ^１およびＹ^２は、水素原子、保護基、または基：

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

を表し；
Ｒ^５およびＲ^６は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択される］
の化合物、そのカチオンラジカル、またはそのジカチオン、またはその塩。
［Ｂ２０］１）α位のアミノ基が保護されたリシンおよびグリセルアルデヒドを反応させて、反応混合物を得ること；
２）反応混合物を分画し、式（Ｉａ）、（Ｉｂ）、（ＩＩａ）」、または（ＩＩｂ）：

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues]
Compound, its cation radical, or its dication, or its salt.
[B20] 1) A reaction mixture is obtained by reacting lysine and glyceraldehyde in which the amino group at the α-position is protected;
2) Fractionate the reaction mixture and formula (Ia), (Ib), (IIa) ”, or (IIb) :.

［式中、Ｒ^１、Ｒ^３、およびＲ^６は、保護基である］
で表される化合物を含む画分を得ること
３）当該画分を用いて動物への免疫を行い、当該画分を抗原とする抗体を得ること
を含む、抗体の製造方法。

[In the formula, R ¹ , R ³ , and R ⁶ are protecting groups]
Obtaining a fraction containing the compound represented by 3) A method for producing an antibody, which comprises immunizing an animal using the fraction to obtain an antibody using the fraction as an antigen.

Among AGEs, glyceraldehyde-derived AGEs (Glycer-AGEs) have been reported to be involved in diabetes, particularly diabetic vascular complications, diabetic retinopathy, diabetic nephropathy, and diabetic macrovascular disease. In addition, Glycer-AGEs include hypertension, dementia such as Alzheimer's disease, cancers (eg liver cancer, pancreatic cancer, uterine cancer, colon cancer, rectal cancer, breast cancer, bladder cancer, malignant melanoma, etc. And lung cancer), non-alcoholic steatosis (NASH), infertility, etc. have been reported to be commonly used. Therefore, Glycer-AGEs can be used as biomarkers for the diagnosis of these diseases, and the antibodies of the present invention can be used in the detection of biomarkers.

Further, the antibody according to the present invention can be used to neutralize the effect of Glycer-AGEs in a living body, and can also be used in the treatment of diseases and the like.

FIG. 1 is a graph showing the results of a reactivity comparison test between a novel monoclonal antibody (SJ-5) and an anti-glyceraldehyde-derived AGEs polyclonal antibody by the ELISA direct method. FIG. 2 is a graph showing the results of a reactivity confirmation test for anti-Glycer-AGEs polyclonal antibody by the ELISA direct method. FIG. 3 is a graph showing the results of a reactivity confirmation test of a POD-labeled SJ-5 antibody by the ELISA direct method. FIG. 4 is a diagram showing the results when GAL13-BSA was used as the immobilized antigen in the confirmation test of the specificity of SJ-5 by the ELISA competition method. FIG. 5 is a diagram showing the results when Glycer-AGEs-BSA was used as the immobilized antigen in the confirmation test of the specificity of the SJ-5 antibody by the ELISA competition method. FIG. 6 is a diagram showing the results of HPLC analysis of Glycer-AGEs-Z-Lys (diode array detection (260 nm)). FIG. 7 is a diagram showing the results of HPLC analysis of Glycer-AGEs-Z-Lys (fluorescence detection (Ex350 nm, Em450 nm)). FIG. 8 is a chart showing the results of mass spectrometry of GAL13. FIG. 9 is a diagram showing the results of measuring electron spin resonance (ESR) with respect to GAL13. FIG. 10 is a graph showing the results of a confirmation test of oxidative activity using 3,3'-diaminobenzidine (DAB) of a sample containing GAL13. FIG. 11 is a graph showing the results of a reactivity evaluation test of SJ-5 antibody and GLAP by competing ELISA. FIG. 12 is a diagram showing the amino acid sequences of the heavy chain variable region and the light chain variable region of SJ-5, which is a novel monoclonal antibody. The underlined part corresponds to the CDR. FIG. 13 is a photograph showing the morphology of HUVEC cultured for 8 hours in the endothelial cell lumen formation suppression test of Example 14. FIG. 14 is a graph showing the expression level of α-SMA in the endothelial mesenchymal conversion inhibition test by Glycer-AGEs-BSA using SJ-5 of Example 15. FIG. 15 is a graph showing the expression level of CD31 in the endothelial mesenchymal conversion inhibition test by Glycer-AGEs-BSA using SJ-5 of Example 15. FIG. 16 shows the LC / MS analysis results of the Glycer-AGEs-Z-Lys reaction solution prepared in Example 16 (diode array detection (260 nm)). FIG. 17 shows the UV spectrum of Glycer-AGEs-Z-Lys prepared in Example 16 at the time of HPLC preparative. FIG. 18 shows the MS spectrum of Glycer-AGEs-Z-Lys prepared in Example 16 at the time of HPLC preparative. FIG. 19 is a graph showing the results of reactivity evaluation of the novel monoclonal antibody of Example 18 by competing ELISA with GAL691 and Lys-hydroxy-triocidin. FIG. 20A is a diagram showing the measurement results of the ESR spectrum of GAL691 performed in Example 19. FIG. 20B is a diagram showing the measurement results of the ESR spectrum of GAL691 supplemented with sodium sulfite. FIG. 20C is a diagram showing the measurement results of the ESR spectrum of GLAP. FIG. 20D is a diagram showing the measurement results of the ESR spectrum of Lys-hydroxy-triocidin. FIG. 21 is a graph showing test results for confirming the effect of white light irradiation on the oxidizing activity of GAL691 on DAB. FIG. 22 is a graph showing the results of a test conducted in Example 21 for confirming the production of singlet oxygen by the photosensitizing action of GAL691. FIG. 23 is a graph showing the results of a test conducted in Example 22 for confirming the production of singlet oxygen by the photosensitizing action of GAL691. FIG. 24 is a graph showing the results of a test conducted in Example 23 for confirming the formation of superoxide anion by the photosensitizing action of GAL691. FIG. 25 is a diagram showing the amino acid sequence of the variable region of the monoclonal antibody PB-1. The underlined part indicates the CDR sequence. FIG. 26A is a graph showing the results of a test for confirming the specificity of the PB-1 antibody when A-peak-BSA was used as the immobilized antigen. FIG. 26B is a graph showing the results of a test for confirming the specificity of the PB-1 antibody when Glycer-AGEs-BSA was used as the immobilized antigen. FIG. 27 is a graph showing the results of a reactivity evaluation test of a novel monoclonal antibody PB-1 with known AGEs by a competing ELISA. FIG. 28 is a graph showing the results of a test for confirming the inhibitory effect of PB-1 antibody on DAB oxidation activity by A-peak-BSA. FIG. 29 is a graph showing the results of a test confirming cross-competition between an SJ-5 antibody and an anti-GA-pyridine monoclonal antibody against a PB-1 antibody. FIG. 30 is a graph showing the results of a tight junction collapse suppression test of retinal pigment epithelial cells (ARPE-19 cells), and shows the results of measuring the impedance of the bottom surface of the well in order to confirm the formation of intercellular tight junctions. It is a graph which shows. FIG. 31 is a photograph showing the morphology of HUVEC cultured for 8 hours in the endothelial cell lumen formation suppression test of Example 35. FIG. 32 is a photograph showing the results of observing the retinal tissue of mice stained with the PB-1 antibody with a confocal laser scanning microscope. FIG. 33 is a diagram showing the results of sample purification using the PB-1 antibody column prepared in Example 37 and analysis of each fraction by electrophoresis (SDS-PAGE). FIG. 34 is a diagram showing the results of Western blotting by electrophoresis (SDS-PAGE) of the protein purified by the PB-1 antibody column prepared in Example 37 and electrotransfer to a PVDF membrane. FIG. 35 is a photograph showing the results of histopathological examination of HE-stained mouse kidney cells. FIG. 36 is a graph showing the binding activity of the PB-1 humanized antibody to the antigen.

In one aspect of the invention, the antibody of the invention is an isolated antibody. The isolated antibody does not include an antibody that is naturally occurring and has not been subjected to any external manipulation (artificial manipulation), that is, an antibody that is produced in the body of an individual and remains there. The antibody of the present invention is typically a monoclonal antibody or an antigen-binding fragment thereof.

［式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択され、
Ｑ^１は、水素原子、または基−ＣＨ_２−Ｘ^１−Ｙ^１を表し；
Ｑ^２は、水素原子、または基−ＣＨ_２−Ｘ^２−Ｙ^２を表し；
Ｘ^１およびＸ^２は、−Ｏ−、または−ＮＨ−を表し；
Ｙ^１およびＹ^２は、水素原子、保護基、または基：

を表し；
Ｒ^５およびＲ^６は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択される］。 In one embodiment of the invention, an antibody that binds to a compound of formula (XI) or (XII), a cation radical thereof, a dication thereof, or a salt thereof is provided:

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
Q ¹ represents a hydrogen atom or group -CH _2- X ^1- Y ¹ ;
Q ² represents a hydrogen atom or group -CH _2- X ^2- Y ² ;
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues].

［式中、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択され、
Ｘ^１およびＸ^２は、−Ｏ−、または−ＮＨ−を表し；
Ｙ^１およびＹ^２は、水素原子、保護基、または基：

を表し；
Ｒ^５およびＲ^６は、それぞれ独立に、水素原子、保護基、およびアミノ酸残基数１〜１０００のペプチド基から選択される］
Ｒ^１、Ｒ^２、Ｒ^３、Ｒ^４、Ｒ^５およびＲ^６が保護基の場合、Ｒ^１、Ｒ^３およびＲ^６はアミノ基の保護基であり、Ｒ^２、Ｒ^４およびＲ^５はヒドロキシ基の保護基である。Ｘ^１またはＸ^２が−Ｏ−の場合、Ｙ^１またはＹ^２はヒドロキシ基の保護基であってもよく、Ｘ^１またはＸ^２が−ＮＨ−の場合、Ｙ^１またはＹ^２はアミノ基の保護基であってもよい。 In another aspect of the invention, an antibody that binds to a compound of formula (I) or (II), a cation radical thereof, a dication thereof, or a salt thereof is provided.

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues]
If R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are protecting groups, then R ¹ , R ³ and R ⁶ are protecting groups for amino groups, and R ² , R ⁴ and R ⁵ are hydroxy. It is a protecting group of the group. If X ¹ or X ² is -O-, then Y ¹ or Y ² may be a hydroxy protecting group, and if X ¹ or X ² is -NH-, then Y ¹ or Y ² is an amino group. It may be a protecting group.

Examples of hydroxy protecting groups include C _1-6 alkyl, C _1-6 alkoxy C _1-6 alkyl, aryl C _1-6 alkyl, heteroaryl C _1-6 alkyl, ((amino C _1-6 alkyl)). Examples thereof include carbonyloxy) C _1-6 alkyl, unsaturated heterocyclic carbonyloxy C _1-6 alkyl, aryl di (C _1-6 alkyl) silyl, tri (C _1-6 alkyl) silyl and the like. Preferred hydroxy protecting groups include C _1-6 alkyl and the like.

Examples of amino protective groups include C _1-6 alkylcarbonyl, aryl C _1-6 alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, C _1-6 alkoxycarbonyl, C _1-6 alkylaminocarbonyl, di (C _{1). -6} alkyl) aminocarbonyl, aryl C _1-6 alkyl, heteroaryl C _1-6 alkyl, (aryl C _1-6 alkyl) aminocarbonyl and the like are included. Preferred amino protecting groups include benzyloxycarbonyl, t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl and the like. In addition, the amino may form a saturated or unsaturated heterocyclic group such as phthalate imide, succinimide, glutarate imide, and 1-pyrrolill by being protected.

The peptide group in the present specification is not particularly limited, and for example, peptide groups having the number of amino acid residues 1 to 10000, 1 to 5000, 1 to 3000, 1 to 2000, 1 to 1000, 1 to 500, and 1 to 100 are used. means. The amino acid residue is selected from natural amino acids.

The compound represented by the formula (I) or (II) can be prepared by using lysine in which the amino group of the side chain is protected. The range represented by the structural formula of the formula (I) or (II) includes the compound represented by the following formula.

In the formula, R ¹ , R ³ , and R ⁶ are amino protecting groups. For example, in the above formula, R ¹ , R ³ , and R ⁶ are benzyloxycarbonyl.
The cationic radicals of the compounds represented by the formulas (I) and (II) are represented by the following formulas (III) and (IV).

In the formulas, R ¹ , R ² , R ³ , R ⁴ , X ¹ , X ² , Y ¹ , and Y ² are as defined in formulas (I) and (II). Formulas (III) and (IV) include cationic radicals represented by the following formulas.

The dications of the compounds represented by the formulas (I) and (II) are represented by the following formulas (V) and (VI).

In the formulas, R ¹ , R ² , R ³ , R ⁴ , X ¹ , X ² , Y ¹ , and Y ² are as defined in formulas (I) and (II). Formulas (V) and (VI) include dications represented by the following formulas.

The salt of the compound represented by the formula (I) or (II) specifically includes a salt formed at the carboxy group of the compound and a salt formed at the peptide group.

The cationic radicals of the compounds represented by the formulas (XI) and (XII) are represented by the following formulas (XIII) and (XIV).

In the formula, R ¹ , R ² , R ³ , R ⁴ , Q ¹ , and Q ² are as defined in formula (XI) and formula (XII).

The dications of the compounds represented by the formulas (XI) and (XII) are represented by the following formulas (XV) and (XVI).

In the formula, R ¹ , R ² , R ³ , R ⁴ , Q ¹ , and Q ² are as defined in formula (XI) and formula (XII).

The salt of the compound represented by the formula (XI) or (XII) specifically includes a salt formed at the carboxy group of the compound and a salt formed at the peptide group. The cation radicals and dications described herein may contain suitable anions.

The term "substantially identical" used with respect to an amino acid sequence means that the sequence difference between the two amino acid sequences being compared is relatively small and the sequence difference has a substantial effect on specific binding to the antigen. It means not to give. Substantially identical amino acid sequences may include modifications of some of the amino acid sequences, eg, deletions, substitutions, or substitutions of one to several (eg, 1-3) amino acids that make up the amino acid sequence. The amino acid sequence may be modified by the addition, insertion, or combination of one to several (for example, 1 to 3) amino acids. The position of the mutation in the amino acid sequence is not particularly limited, and the mutation may occur at a plurality of positions. The number of amino acids modified in the amino acid sequence is, for example, within 10% of the total amino acids shown, preferably within 5% of the total amino acids. More preferably, it is a number corresponding to within 1% of all amino acids.

When substituting an amino acid, it can be replaced with an amino acid having a side chain having similar biochemical properties to the side chain of the amino acid to be replaced (conservative amino acid substitution). In one embodiment, the substantially identical amino acid sequences may include, for example, one or more conservative substitutions. Conservative amino acid substitutions are known to those of skill in the art and include, for example, the examples shown in the table below (eg, WO2010 / 146550, etc.).

In one embodiment, the two amino acid sequences having 80% or more, 85% or more, 90% or more or 95% or more identity are substantially identical. In another embodiment, the two amino acid sequences having 80% or more, 85% or more, 90% or more or 95% or more identity use the amino acids whose substitutions are shown in the conservative or exemplary substitutions shown in the table above. When replaced, they are substantially the same. In yet another embodiment, the two amino acid sequences having 80% or more, 85% or more, 90% or more or 95% or more identity are substituted with the amino acids shown in the conservative substitutions in the table above. If so, they are substantially the same.

In addition, naturally occurring amino acids are classified into the following groups based on common side chain properties:
(1) Non-polarity: norleucine, Met, Ala, Val, Leu, Ile,
(2) Polarity, no charge: Cys, Ser, Thr, Asn, Gln,
(3) Acid (load electricity): Asp, Glu,
(4) Basic (positive charge): Lys, Arg, His,
(5) Residues affecting chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe.

As one embodiment, amino acids belonging to the same group as the amino acids to be substituted may be used for substitution.

In one embodiment, the two amino acid sequences having 80% or more, 85% or more, 90% or more or 95% or more identity are substantially identical. In another embodiment, two amino acid sequences having 80% or more, 85% or more, 90% or more or 95% or more identity are substituted with amino acids and amino acids belonging to the same group as the above group. If so, they are substantially the same.

Whether or not the two amino acid sequences are substantially the same can be determined by comparing the binding specificity of the antibody containing each amino acid sequence (the sequences of the other regions are the same) to the antigen. _{For example, when the dissociation constant (K d} value) of the reference antibody for the antigen in the physiological saline environment is set to A, the K _d value of the antibody to be compared is in the range of A × 10 ^{-1 to} A × 10. If so, the substantial identity can be recognized.

The nucleic acid according to the present invention is typically an isolated nucleic acid, and in the case of a nucleic acid produced by a gene recombination technique such as a cDNA molecule, the "isolated nucleic acid" is preferably a cell component or a culture. Nucleic acid in a state that does not substantially contain liquid or the like. Similarly, in the case of nucleic acids produced by chemical synthesis, the "isolated nucleic acid" is preferably in a state in which it is substantially free of precursors (raw materials) such as dNTPs and chemical substances used in the synthetic process. Refers to nucleic acid.

As used herein, the term "nucleic acid" includes DNA (including cDNA and genomic DNA), RNA (including mRNA), DNA analogs, and RNA analogs. The form of the nucleic acid of the present invention is not limited, that is, it may be either single-stranded or double-stranded. It is preferably double-stranded DNA. Codon degeneration is also considered. That is, in the case of a nucleic acid encoding a protein, it may have an arbitrary base sequence as long as the protein can be obtained as an expression product thereof.

AGEs are formed by the glycation reaction of proteins. Reducing sugars such as glucose and fructose react non-enzymatically with free amino groups of proteins to produce Amadori compounds from Schiff bases, and then irreversible reactions such as dehydration, condensation, oxidation, and reduction are repeated, which is peculiar. AGEs, a complex yellow-brown substance with fluorescence, are produced. On the other hand, compounds obtained by reacting amino acid residues such as lysine with sugar have been identified as the structure of AGEs, and have no fluorescence, pyrarin, N ^ε -carboxymethyl lysine (CML), and N ^ε -carboxyethyl. Lysine (CEL), N ^ω -carboxymethyl arginine (CMA) and the like are also known as AGEs. In addition, compounds having an aromatic ring such as argpyrimidine, pentosidine, crosulin, GA-pyridine, vesperurisine, and pyropyridine have been specified as AGEs. Peptides and proteins containing these compounds as amino acid residues are also included in AGEs.

As substrates for protein glycation reactions that form AGEs, in addition to reducing sugars such as glucose and fructose, glyceraldehyde, glycolaldehyde, methylglyoxal, glyoxal, and 3-deoxyglucosone, which are produced by sugar metabolism in the living body, are used. Son etc. are assumed. Attempts have been made to use these reducing sugars and aldehydes to react with proteins such as BSA to form AGEs. Glyceraldehyde-derived AGEs (Glycer-AGEs), glucose-derived AGEs (Glu-AGEs), glycolaldehyde-derived AGEs (Glycol-AGEs), fructose-derived AGEs (Fru-AGEs), and methylglioxal-derived AGEs prepared in this manner. Among various AGEs such as (MGO-AGEs), glycal-derived AGEs (GO-AGEs), and 3-deoxyglucosone-derived AGEs (3-DG-AGEs), glyceraldehyde-derived AGEs are used for various diseases such as diabetes. It has been reported that it is involved, and a method for measuring the blood concentration of Glycer-AGEs by an analysis method using a polyclonal antibody using AGEs derived from glyceraldehyde as an antigen is known.

The monoclonal antibody of the present invention is obtained by fractionating AGEs (Glycer-AGEs-Z-Lys) obtained by reacting lysine in which the amino group at the α-position is protected with a protecting group (Z group) with glyceraldehyde. The specific fraction to be used can be prepared as an antigen. Further, the monoclonal antibody of the present invention has a property of specifically binding to glyceraldehyde-derived AGEs (Glycer-AGEs) and glycolaldehyde-derived AGEs (Glycol-AGEs).

In one aspect of the invention, it binds to epitopes of glyceraldehyde-derived AGEs (Glycer-AGEs) and / or glycolaldehyde-derived AGEs (Glycol-AGEs), glucose-derived AGEs (Glu-AGEs), and fructose-derived AGEs (Fru). -AGEs), methylglyoxal-derived AGEs (MGO-AGEs), glyoxal-derived AGEs (GO-AGEs), and 3-deoxyglucosone-derived AGEs (3-DG-AGEs) that bind to one or more AGEs. No, a monoclonal antibody or an antigen-binding fragment thereof is provided. More specifically, it binds to epitopes of glyceraldehyde-derived AGEs (Glycer-AGEs) and glycolaldehyde-derived AGEs (Glycol-AGEs), and glucose-derived AGEs (Glu-AGEs), fructose-derived AGEs (Fru-AGEs), and the like. Provided are monoclonal antibodies or antigen-binding fragments thereof that do not bind to methyl glyoxal-derived AGEs (MGO-AGEs), glyoxal-derived AGEs (GO-AGEs), and 3-deoxyglucosone-derived AGEs (3-DG-AGEs). .. More specifically, AGEs derived from glyceraldehyde and AGEs derived from glycol aldehyde bind to epitopes of AGEs derived from glucose, AGEs derived from glucose (Glu-AGEs), AGEs derived from fructose (Fru-AGEs), AGEs derived from methyl glyoxal (MGO-AGEs), and the like. Monochrome that does not bind to AGEs derived from glyoxal (GO-AGEs), AGEs derived from 3-deoxyglucoson (3-DG-AGEs), N ^ε -carboxymethyl lysine (CML), and N ^ε -carboxyethyl lysine (CEL). An antibody or an antigen-binding fragment thereof is provided.

In one aspect of the invention, a monoclonal antibody or antigen-binding fragment thereof that binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs is provided. Here, glyceraldehyde-derived AGEs and glycolaldehyde-derived AGEs are AGEs produced by protein saccharification reactions such as BSA and mouse serum albumin (MSA) in the presence of glyceraldehyde or glycolaldehyde, and are proteins containing AGEs. .. The monoclonal antibody of the present invention and its antigen-binding fragment have a feature that the chemical structure generated in the protein saccharification reaction is used as an epitope. In a preferred embodiment, the antibody of the present invention and an antigen-binding fragment thereof are derived from aldehydes generated by reduced sugar and sugar metabolism, particularly glucose-derived AGEs, fructose-derived AGEs, methylglyoxal-derived AGEs, glyoxal-derived AGEs, and 3-deoxyglucosone. It does not combine with AGEs. These AGEs are also prepared by a protein saccharification reaction by adding each reducing sugar or aldehyde to a protein such as BSA. The binding specificity of an antibody can be specified by a known method, such as a competitive ELISA method.

The present invention provides monoclonal antibodies or antigen-binding fragments thereof that bind to epitopes of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs, and the antibodies or fragments are used for the treatment, prevention and / or diagnosis of diseases associated with AGEs. Can be used. The antibodies that can be used in the present invention can take multiple forms as described herein, and the present invention is a set of 6 CDRs (substantially identical amino acids) as defined herein. Provided is an antibody structure comprising a sequence, eg, a sequence comprising deletions, substitutions, or additions of 1-3 amino acid residues.

Conventional antibody structural units typically contain tetramers. Each tetramer typically consists of two identical polypeptide chain pairs, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy". It has a chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified into κ light chains and λ light chains. Heavy chains are classified as μ, δ, γ, α or ε and define antibody isotypes as IgM, IgD, IgG, IgA and IgE, respectively. IgG has a plurality of subclasses, and subclasses include, but are not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but is not limited to, IgM1 and IgM2. Thus, as used herein, "isotype" means any subclass of immunoglobulin defined by the chemical and antigenic characteristics of its constant region. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE. Therapeutic antibodies may also include hybrids of isotypes and / or subclasses. In one embodiment, the antibody of the invention is IgG, IgA, IgM, IgD or IgE, preferably IgG.

Each strand contains variable regions of about 100-110 or more amino acids that are primarily involved in antigen recognition. In the variable region, three loops are assembled for each V domain of heavy chain and light chain to form an antigen binding site. Each loop is also referred to as a complementarity determining region (hereinafter, also referred to as "CDR"), and the variation of the amino acid sequence in this region is most prominent. By "variable" is meant the fact that a particular segment of the variable region varies widely in the sequence of the antibody. The variability within the variable region is not evenly distributed. Instead, the V region is referred to as the framework region (FR) of 15-30 amino acids, each separated by an extremely shortly variable region called a "hypervariable region" that is 9 to 15 amino acids or more in length. It consists of a relatively immutable stretch.

Each VH and VL consists of three hypervariable regions (“complementarity determining regions”, “CDRs”) and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4.

The hypervariable regions are generally around amino acid residues 24-34 (VL CDR1; "VL" means variable region of the light chain), around 50-56 (VL CDR2) and 89-97 in the light chain variable region. Near (VL CDR3) and heavy chain variable region, around 31-35 (VH CDR1; "VH" means variable heavy chain region), around 50-65 (VH CDR2) and around 95-102 (VH CDR3) ) Amino acid residues; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and / or those forming a hypervariable loop. Residues (eg, residues 26-32 (VL CDR1), 50-52 (VL CDR2) and 91-96 (VL CDR3) in the light chain variable region and 26-32 (VH CDR1) in the heavy chain variable region, Includes 53-55 (VH CDR2) and 96-101 (VH CDR3); Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917).

The EU number used in the Fc region is used herein to refer to variables in the variable domain (approximately residues 1-107 in the light chain variable region and residues 1-113 in the heavy chain variable region). A Kabat numbering system is commonly used with the system (eg, Kabat et al., Supra (1991)). The identification of the CDR by Kabat numbering can be specified by using generally available software (for example, abYsis (http://www.abysis.org/)).

CDRs contribute to the formation of antigen binding, and more specifically to the formation of epitope binding sites for antibodies. "Epitope" means a determinant that interacts with a specific antigen binding site (paratope) in the variable region of an antibody molecule. Epitopes are grouped into molecules such as amino acids and sugar side chains and usually have specific structural and specific charge properties. As shown herein, the antibody referred to herein as "SJ-5" or "PB-1" according to the present invention is the above formula (XI) or (XII), or the above formula (I) or (II). ) Is considered to bind to the antigen as an epitope.

Various AGEs can be prepared by mixing proteins (for example, bovine serum albumin (BSA), mouse serum albumin (MSA), rabbit serum albumin (RSA), etc.) with aldehydes or reducing sugars and reacting them. For example, by reacting glyceraldehyde with BSA, BSA containing AGEs derived from glyceraldehyde can be prepared, and this can be used as a standard for AGEs. In glyceraldehyde-derived AGEs, the antibody of the present invention is prepared, for example, by selecting an antibody having binding property to glyceraldehyde-derived AGEs-containing BSA and not binding to BSA. be able to. In one aspect of the invention, the epitope to which the antibody of the invention binds comprises a structure produced by a non-enzymatic reaction between glyceraldehyde and a protein.

In one aspect of the present invention, an antibody containing the amino acid sequences specified in (1-1) and (1-2) below in the complementarity determining regions of the heavy chain variable region and the light chain variable region, or antigen binding thereof. Fragments provided:
(1-1)
(A) The amino acid sequence shown in VH CDR1: SEQ ID NO: 1 or an amino acid sequence substantially the same as that;
(B) VH CDR2: Amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence substantially the same as that;
(C) VH CDR3: Amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence substantially the same as that;
(D) VL CDR1: The amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence substantially the same as that;
(E) VL CDR2: Amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence substantially the same as that; and (f) VL CDR3: Amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence substantially the same as the amino acid sequence. Or (1-2)
(G) VH CDR1: The amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence substantially the same as that;
(H) VH CDR2: Amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence substantially the same as that;
(I) VH CDR3: Amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence substantially the same as that;
(J) VL CDR1: The amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence substantially the same as that;
(K) VL CDR2: Amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence substantially the same as that;
(L) VL CDR3: Amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence substantially the same as that.

According to one aspect of the present invention, the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 4 or substantially the same amino acid sequence, and the amino acid sequence of the light chain variable region is SEQ ID NO: 8. Provided is a monoclonal antibody or an antigen-binding fragment thereof, which has an amino acid sequence of the above or an amino acid sequence substantially the same as that of the amino acid. As a specific embodiment thereof, the present invention includes SJ-5, which is a monoclonal antibody described in the present specification. According to another aspect of the present invention, the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO: 18 or substantially the same amino acid sequence, and the amino acid sequence of the light chain variable region is SEQ ID NO: 22. Provided is a monoclonal antibody or an antigen-binding fragment thereof, which has an amino acid sequence of the above or an amino acid sequence substantially the same as that of the amino acid. As a specific embodiment, the present invention includes PB-1, which is a monoclonal antibody described herein.

According to one aspect of the invention, these antibodies are mouse-derived antibodies and have constant regions of the mouse antibody in addition to the heavy and light chain variable regions set forth in the above SEQ ID NOs. There is. An example of an amino acid sequence substantially the same as the heavy chain variable region or the light chain variable region shown by the above SEQ ID NO: is a sequence in which one or two amino acids are added to the N-terminal or C-terminal. .. For example, as an amino acid sequence substantially the same as the heavy chain variable region represented by the above SEQ ID NO:, one or two amino acids at the N-terminal or C-terminal (for example, an amino acid selected from natural amino acids), more specifically. Includes a sequence to which one amino acid selected from Glu and Gln is added.

According to one aspect of the present invention, glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs are referred to as the above-mentioned heavy chain variable region and light chain variable region or the antibody identified by their CDRs or an antigen-binding fragment thereof as a reference antibody. An antibody that cross-competes with a reference antibody or an antigen-binding fragment thereof is provided for binding to an epitope of. In one embodiment of the present invention, an antibody or an antigen-binding fragment thereof that cross-competes with the reference antibody for binding to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs, using the monoclonal antibody SJ-5 as a reference antibody. Provided.

The antibody of the present invention can be confirmed to have cross-competitive properties by performing a standard binding assay with Glycer-AGEs or Glycol-AGEs, for example, using the monoclonal antibody SJ-5 as a reference antibody. Confirmation of cross-competition can be confirmed, for example, by flow cytometry or cross-competition ELISA assay.

In one aspect of the invention, a pharmaceutical composition for treating, preventing or diagnosing a disease is provided that comprises the antibody or antigen-binding fragment thereof. The target disease is not particularly limited as long as it is a disease involving Glycer-AGEs or Glycol-AGEs, and includes, for example, diabetes, diabetic minimal vascular complications (diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy), and diabetic neuropathy. Dementia such as diabetic macroangiopathy, hypertension, Alzheimer's disease, cancers (eg liver cancer, pancreatic cancer, uterine cancer, colon cancer, rectal cancer, breast cancer, bladder cancer, malignant melanoma, and Lung cancer), non-alcoholic steatosis (NASH), infertility and the like. According to another aspect of the invention, the disease is diabetes, impaired glucose tolerance, retinopathy, nephropathy, peripheral neuropathy, lower extremity necrosis, arteriosclerosis, thrombosis, non-alcoholic steatohepatopathy, non-alcoholic fat. These include hepatitis, cancer (eg, melanoma, lung cancer, and liver cancer), polycystic ovary syndrome, ovarian dysfunction, central nervous system disorder, and Alzheimer's disease.

In one aspect of the invention, the pharmaceutical compositions of the invention can be used for the treatment, prevention or diagnosis of diseases caused by endothelial mesenchymal conversion promoted by AGEs. Target diseases include cancer (for example, ovarian cancer, breast cancer, uterine body cancer, prostate cancer, tongue cancer, oral cancer, pharyngeal cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer). , Pancreatic cancer, lung cancer, liver cancer, brain tumor, etc.), obesity, diabetes, diabetic diseases (eg, diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic myocardium, diabetic vascular complications, diabetic nephropathy, Diabetic kidney disease, diabetic foot lesions, diabetic ketoacidosis, diabetic nephropathy, etc.), periodontal disease, age-related luteal degeneration, lung disease or respiratory disease (eg, pulmonary fibrosis, idiopathic pulmonary fibrosis, fine Peribronchial fibrosis, interstitial lung disease, cancer fibrous lung disease, chronic obstructive lung disease, peripheral airway disease, pulmonary emphysema, etc.), renal disease (renal nephritis, glomerulosclerosis, glomerulonephritis, mesangial proliferative thread) (Spheroid nephritis, renal systemic fibrosis, renal interstitial fibrosis, chronic kidney disease, etc.), idiopathic retroperitoneal fibrosis, scleroderma, female infertility, polycystic ovary syndrome, ovarian insufficiency, early ovarian function Insufficiency, male infertility, liver disease (eg liver cirrhosis, non-alcoholic steatosis, etc.), cardiovascular disease (eg acute lower extremity arterial embolism, peripheral arterial disease, atherosclerotic cerebral infarction, atherosclerosis, internal Carotid artery stenosis, aortic valve stenosis, aortic valve insufficiency, angina, congestive heart failure, acute heart failure, chronic heart failure, ischemic heart disease, dilated cardiomyopathy, cardiac sarcoidosis, hypertension, pulmonary arterial pulmonary hypertension , Pulmonary heart, myocarditis, vascular stenosis heart fibrosis, post-myocardial infarction cardiac fibrosis, post-myocardial infarction left ventricular hypertrophy, cerebral infarction, cerebrovascular disorder, etc.), rheumatoid arthritis, lifestyle disease, dyslipidemia, Alzheimer's disease , Vascular dementia, vaginitis, endocrine disorders, osteoporosis. More specifically, diabetic retinopathy, diabetic nephropathy, atherosclerotic cerebral infarction, atherosclerosis, chronic kidney disease, chronic heart failure, and ischemic heart disease are exemplified.

The pharmaceutical compositions of the present invention can be used for the diagnosis, treatment and / or prevention of eye diseases. In one embodiment, the pharmaceutical composition can be used to confirm the degree of progression of an eye disease or to estimate the risk of developing an eye disease. In another embodiment, the pharmaceutical composition can be used in a treatment to prevent or delay the progression of an eye disease that has developed, eg, can be administered regularly over a long period of time. In yet another embodiment, the pharmaceutical composition can be prophylactically administered to a subject at high risk of developing the disease. In yet another embodiment, the pharmaceutical composition can be used in the treatment of developing an eye disease to restore the site of onset of the disease.

Suitable eye diseases for use of the pharmaceutical composition of the present invention include, for example, diabetic retinopathy, diabetic cataract, retinal pigment degeneration, diabetic macular edema, Labor congenital macular disorder, Stargart's disease, Asher syndrome, colloideremia, rod cone. Body dystrophy, pyramidal dystrophy, progressive retinal atrophy, age-related macular degeneration, macular dystrophy, choriosclerosis, total chorioretinal atrophy, cystic macular edema, vasculitis, retinal exfoliation, macular foramen, macular capillary Includes vasodilation, macula, optic neuropathy, ischemic retinal disease, premature infant retina, retinal vascular occlusion, and retinal aneurysm. Suitable eye diseases include, for example, diabetic retinopathy, diabetic macular edema, retinitis pigmentosa and age-related macular degeneration.

The pharmaceutical composition of the present invention has the effect of reducing or eliminating the inhibitory effect of AGEs on the lumen formation of vascular endothelial cells, and further suppressing or inhibiting the endothelial mesenchymal conversion of vascular endothelial cells induced by AGEs. Due to such an effect, the increase in vascular permeability due to inhibition of lumen formation is reduced or eliminated, and cancer metastasis is suppressed. Therefore, in one embodiment, the pharmaceutical composition of the present invention can be used for the prevention or suppression of cancer metastasis.

One aspect of the invention comprises the step of measuring the amount of Glycer-AGEs and Glycol-AGEs present in a tissue of interest (eg, blood) using an antibody of the invention or an antigen-binding fragment thereof. A diagnostic method is provided. The diagnosis here is as described above. In another aspect of the present invention, a step of measuring the amount of Glycer-AGEs and Glycol-AGEs present in a sample (for example, a blood sample obtained by blood sampling) using the antibody of the present invention or an antigen-binding fragment thereof. Methods for analyzing the sample, including, are provided.

The antigen-binding fragment of the antibody of the present invention is, for example, Fab, Fab', F (ab') ₂ , Fv, scFv, dsFv, diabody, or sc (Fv) ₂ . The antibody of the present invention is, for example, a mouse antibody, a humanized antibody, a human antibody, or a chimeric antibody. These antibodies or antigen-binding fragments thereof can be prepared by methods known to those skilled in the art.

The antigen-binding fragment of the antibody of the present invention is, for example, 1 × 10 ^-4 M or less, specifically 1 × 10 ^-5 M or less, more specifically 1 × 10 ^-6 M or less, and more specifically, 1 × 10 -6 M or less. It binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs with an equilibrium dissociation constant _Kd of 1 × ^10-7 M or less, preferably 1 × ^{10-8 M or less.} The _Kd value of an antibody can be determined using well-established methods in the art. _{A preferred method for determining the Kd} value of an antibody is by using surface plasmon resonance, preferably by using a biosensor system such as the Biacore® system.

In one aspect of the present invention, there is provided a method for producing a monoclonal antibody or an antigen-binding fragment thereof that binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs. The method comprises culturing a host cell transfected with a nucleic acid encoding an antibody of the invention and can be carried out in a variety of ways based on well-known techniques.

In one aspect of the invention, nucleic acids encoding the antibodies of the invention are provided. Such polynucleotides encode, for example, both variable and constant regions of the heavy and light chains, respectively. The polynucleotide may be in the form of RNA or DNA. The coding sequence encoding the polypeptide may include genetic code redundancy or degeneracy.

In some embodiments, one or more nucleic acids encoding the antibodies of the invention are integrated into an expression vector, which expression vector is extrachromosomal or integrated into the genome of the host cell into which it is introduced. Can be designed to be. Expression vectors include any number of suitable regulatory sequences, including, but not limited to, transcriptional and translational regulatory sequences, promoters, ribosome binding sites, enhancers, origins of replication, or other elements (such as selected genes). All of these can be operably linked as is well known in the art. In some cases, two nucleic acids are used and each is placed in a different expression vector (eg, a nucleic acid encoding a heavy chain in a first expression vector, a nucleic acid encoding a light chain in a second expression vector). Alternatively, they can be placed in the same expression vector. The design of one or more expression vectors, including the selection of control sequences, can be determined by factors such as host cell selection, desired protein expression level, and the like.

In general, one or more nucleic acids can be manipulated into one or more expression regulators using any method suitable for the host cell of choice (eg, transformation, transfection, electroporation, infection, etc.). Recombinant host cells are introduced into host cells of suitable nucleic acid and / or expression so as to be ligated (eg, in a vector, integrated into the genome of the host cell in a construct created by a process in the cell). Is created. The obtained recombinant host cell is prepared under conditions suitable for expression (for example, in the presence of an inducer, in a suitable non-human animal, a medium supplemented with a suitable salt, growth factor, antibiotic, dietary supplement, etc. ), Which produces one or more encoded polypeptides. In some cases, the heavy chain is made in one cell and the light chain is made in another cell.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), Manassas, VA. Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human liver cancer cells (eg Hep G2), and many others Cell lines include, but are not limited to. Recombinant antibodies can also be expressed using non-mammalian cells, including but not limited to bacteria, yeasts, insects and plants. In some embodiments, the antibody can be produced in transgenic animals such as cows and chickens.

In one aspect of the present invention, there is provided a method for producing an antibody by immunizing an animal using a compound of formula (XI) or formula (XII), or a compound of formula (I) or formula (II) as an antigen. .. The method for producing the compound of the formula (XI) or the formula (XII), or the compound of the formula (I) or the formula (II) is not particularly limited, and for example, lysine and glyceraldehyde in which the amino group at the α-position is protected are reacted. The reaction mixture thus obtained can be purified and prepared. As the antigen used for immunization, for example, among the fractions obtained by fractionating the above reaction mixture, a fraction containing a compound represented by the formula (Ia) or the formula (Ib) can be used. Fractionation can be performed by a conventional method, for example, a fraction containing a specific peak can be used by HPLC fractionation.

Immunization can use the methods commonly used in the production of antibodies. As the animal, mammals other than humans such as mice, rats, rabbits, dogs, pigs, hamsters and the like can be used.

[Example 1] Preparation of AGEs AGEs can be prepared by a known method (Non-Patent Document 5 and Non-Patent Document 6 and the like). Specifically, various AGEs were prepared by the following methods.

(1) Bovine serum albumin containing AGEs derived from glyceraldehyde (Glycer-AGEs-BSA)
Bovine serum albumin (BSA, Sigma-Aldrich), DL-glyceraldehyde (DL-GLA, Nacalai Tesque), diethylenetriamine-N, N, N', N ", N" -pentaacetic acid (DTPA, Dojin Chemical Research Institute) Was obtained by purchase.

DL-GLA (180 mg) and DTPA (39 mg) were weighed and transferred to a 50 mL culture tube. Phosphate buffer (0.2 M, pH 7.4, 20 mL) was added to the tube, and the mixture was stirred with a vortex mixer until DL-GLA was dissolved. Then, BSA (500 mg) was added and stirred until dissolved (concentration of BSA in solution: 25 mg / mL).

The resulting mixture was passed through a 0.2 μm filter in a clean bench to give a sterile solution. The tube lid was sealed with a film such as Parafilm ™ to prevent the liquid in the tube from evaporating, and the tube was incubated at 37 ° C. for 1 week.

The resulting reaction solution (2.5 mL each) was applied to a PD-10 column (GE Healthcare, 8 bottles) and according to the PD-10 column protocol, phosphate buffered saline (PBS) (pH 7. It was eluted with 4 and 3.5 mL each).

Eluate for 4 columns (14 mL in total) was collected and placed in a dialysis tube (Spectra / Por Dialysis Membrane, width: 23 mm) with a molecular weight cut-off of 6 to 8 kDa. It was placed in PBS, stirred and dialyzed. Dialysis was performed in a low temperature room (5 ° C.), the dialysate (PBS) was changed every 24 hours, and dialysis was performed for 3 days.

After the completion of dialysis, the solutions in the two dialysis tubes were put together into a 50 mL culture tube, the amount of protein was measured as glyceraldehyde-derived AGEs-containing BSA (Glycer-AGEs-BSA), and the culture PBS was used. Adjusted to the required protein concentration.
A control BSA was prepared in the same manner as in Example 1 except that DL-GLA was not added.

(2) Glyceraldehyde-derived AGEs-containing mouse serum albumin (Glycer-AGEs-MSA) and glyceraldehyde-derived AGEs-containing rabbit serum albumin (Glycer-AGEs-RSA)
25 mg / mL mouse serum albumin (MSA, Sigma-Aldrich) or rabbit serum albumin (RSA, Sigma-Aldrich), DL-glyceraldehyde (0.1 M) in 0.2 M phosphate buffer (pH 7.4). , Nakaraitesk), and diethylenetriamine-5acetic acid (5 mM, DTPA, Dojin Chemical Laboratory). The solution was sterilized by 0.2 μm filter sterilization and incubated at 37 ° C. for 1 week. Low molecular weight unreacted substances are removed using a PD-10 gel filtration column (GE Healthcare), and further dialyzed in PBS (phosphate buffered saline) for 3 days in a low temperature room (5 ° C.) (during this period). , Change PBS daily).

(3) Glucose-derived AGEs-containing BSA (Glu-AGEs-BSA) and fructose-derived AGEs-containing BSA (Fru-AGEs-BSA)
25 mg / mL bovine serum albumin (BSA, Sigma-Aldrich), D-glucose (0.5 M, Wako Pure Chemical Industries, Ltd.) or D-fluctose (0.5 M, 0.5 M,) in 0.2 M phosphate buffer (pH 7.4). A solution containing Wako Pure Chemical Industries, Ltd.) and DTPA (5 mM, Dojin Chemical Industries, Ltd.) was prepared, and the solution was sterilized by 0.2 μm filter sterilization and incubated at 37 ° C. for 8 weeks. Low molecular weight unreacted substances were removed using a PD-10 gel filtration column, and further dialyzed with PBS in a low temperature room (5 ° C.) for 3 days (PBS was replaced daily during this period).

(4) Glycolaldehyde-derived AGEs-containing BSA (Glycol-AGEs-BSA), methylglyoxal-derived AGEs-containing BSA (MGO-AGEs-BSA), and glyoxal-derived AGEs-containing BSA (GO-AGEs-BSA).
0.1 M glycolaldehyde (Sigma-Aldrich), methylglyoxal (Sigma-Aldrich), or glyoxal (Sigma-Aldrich) and BSA (25 mg / mL) in 0.2 M phosphate buffer (pH 7.4). And a solution containing DTPA (5 mM) was prepared. The solution was sterilized by 0.2 μm filter sterilization and incubated at 37 ° C. for 1 week. Incubate at 37 ° C. for 1 week under aseptic conditions, then remove low molecular weight unreacted substances using a PD-10 gel filtration column, and further dialyze with PBS in a low temperature room (5 ° C.) for 3 days (during this period). , Change PBS daily).

(5) 3-Deoxyglucosone-derived AGEs-containing BSA (3-DG-AGEs-BSA)
Prepare a solution containing BSA (25 mg / mL, Sigma-Aldrich), 3-deoxyglucosone (0.2 M, Dojin Chemical Laboratory) and DTPA (5 mM) in 0.2 M phosphate buffer (pH 7.4). bottom. The solution was sterilized by 0.2 μm filter sterilization and incubated at 37 ° C. for 2 weeks. Then, low molecular weight unreacted substances and the like were removed using a PD-10 gel filtration column, and further dialyzed with PBS in a low temperature room (5 ° C.) for 3 days (PBS was replaced daily during this period).

(6) N ^ε -carboxymethyl lysine-containing BSA (CML-BSA)
BSA (50 mg / mL, Sigma-Aldrich), glyoxylic acid (45 mM, Wako Pure Chemical Industries, Ltd.) and sodium cyanoborohydride (150 mM, Sigma-Aldrich) in 0.2 M phosphate buffer (pH 7.4). A solution containing was prepared. The solution was sterilized by 0.2 μm filter sterilization and incubated at 37 ° C. for 24 hours. Then, low molecular weight unreacted substances and the like were removed using a PD-10 gel filtration column, and further dialyzed with PBS in a low temperature room (5 ° C.) for 3 days (PBS was replaced daily during this period).

(7) N ^ε -carboxyethyl lysine-containing BSA (CEL-BSA)
BSA (50 mg / mL, Sigma-Aldrich), pyruvic acid (45 mM, Wako Pure Chemical Industries, Ltd.) and sodium cyanoborohydride (150 mM, Sigma-Aldrich) in 0.2 M phosphate buffer (pH 7.4). A solution containing was prepared. The solution was sterilized by 0.2 μm filter sterilization and incubated at 37 ° C. for 24 hours. Then, low molecular weight unreacted substances and the like were removed using a PD-10 gel filtration column, and further dialyzed with PBS in a low temperature room (5 ° C.) for 3 days (PBS was replaced daily during this period).

[Example 2] Preparation of antigen (1) glyceraldehyde-derived AGEs lysine (Glycer-AGEs-Z-Lys ) Preparation of N ^alpha - carbobenzoxy -L- lysine (Z-Lys-OH, Tokyo Kasei Kogyo) , DL-glyceraldehyde (DL-GLA, Nacalai Tesque) was obtained by purchase. DL-GLA (675.6 mg) was weighed and transferred to a 50 mL centrifuge tube. Phosphate buffer (0.2 M, pH 7.4, 25 mL) was added to the centrifuge tube, and the mixture was stirred with a vortex mixer until DL-GLA was dissolved. Then, Z-Lys-OH (700.8 mg) was added and stirred until dissolved (DL-GLA concentration in solution: 300 mM, Z-Lys-OH concentration: 100 mM). Then, the lid of the tube was sealed with a film such as Parafilm (trademark) so that the liquid in the centrifuge tube would not evaporate, and the tube was allowed to stand at 37 ° C. for 1 week or longer.

To 5 mL of the obtained reaction solution, 1 mL of a 10% trifluoroacetic acid (TFA, Wako Pure Chemical Industries, Ltd.) aqueous solution was added and mixed by inversion. Then, it was centrifuged (12,000 × g) for 10 minutes at room temperature, and the supernatant was removed. Ultrapure water (5 mL) was added to the centrifuge tube containing the obtained precipitate, and centrifugation was performed again to remove the supernatant. The precipitate was washed by performing this operation a total of three times. The precipitate after washing was air-dried, the dry weight was measured (200 mg), and the precipitate was stored in a refrigerator.

(2) Precipitating and purifying Glycer-AGEs-Z-Lys The precipitate of Glycer-AGEs-Z-Lys was dissolved in a phosphate buffer solution (0.2M, pH 7.4) and 0.5 mg / mL (for analysis). ) Or 50 mg / mL (for precipitation) sample solution was prepared. Then, the mixture was filtered through a syringe filter (Millex-LG, Merck) having a pore size of 0.2 μm, and the filtrate was used as a sample for high performance liquid chromatography (HPLC). The HPLC instrument used the 1260 Infinity II system (Agilent Technologies) and consisted of the following modules; quarterly pump (G7111B), multisampler (G7167A), multicolumn thermostat (G7116A), diode array detector (G7115A). , Fluorescence detector (G7121B), Fraction collector (G1364F), OpenLAB CDS ChemStation software. The mobile phase was prepared from the HPLC solvent of Wako Pure Chemical Industries, Ltd. The mobile phase A was a 10 mM ammonium acetate aqueous solution obtained by mixing 10 mL of a 1 M ammonium acetate solution and 990 mL of distilled water, and the mobile phase B was acetonitrile. The analysis time was 0-10 minutes flowing down at mobile phase A: B = 75: 25, 10.1-20 minutes flowing down at mobile phase A: B = 65:35, and 20.1-2 minutes flowing down at mobile phase A. : B = 10: 90 flowed down. A YMC-Triart C18 (150 × 4.6 mm, YMC) was used as the analysis column, and the flow rate was set to 0.8 mL / min. 5 μL of the sample solution for analysis (0.5 mg / mL) after filtration was injected into the HPLC apparatus, and Glycer-AGEs-Z-Lys was used with a diode array detector (260 nm) and a fluorescence detector (excitation wavelength 350 nm, fluorescence wavelength 450 nm). Peak was detected. The results of HPLC are shown in FIG. 6 (diode array detection (260 nm)) and FIG. 7 (fluorescence detection (Ex350 nm, Em450 nm)).

As a result, a remarkable peak was observed at the analysis time of 13.5 minutes. Hereinafter, this peak will be referred to as GAL13. Subsequently, YMC-Triart C18 (150 × 10 mm, YMC) was attached to the HPLC apparatus, and the flow rate was set to 2.5 mL / min. 50 μL of the filtered sample solution (50 mg / mL) was injected into the HPLC apparatus, and GAL13 was detected by a diode array detector (260 nm). Then, GAL13 was separated by a fraction collector set with a UV peak as a trigger.

Acetonitrile contained in the preparative solution was removed by a rotary evaporator (Tokyo Rika Kikai). To 5 mL of the residue solution, 1 mL of a 10% TFA aqueous solution was added and mixed by inversion. Then, it was centrifuged (12,000 × g) for 10 minutes at room temperature, and the supernatant was removed. Ultrapure water (5 mL) was added to the obtained precipitate, and centrifugation was performed again to remove the supernatant. The precipitate was washed by performing this operation a total of three times. The precipitate after washing was air-dried, the dry weight was measured (1 mg), and the precipitate was stored in a refrigerator.

(3) Binding of GAL13 to carrier protein The dry powder of GAL13 after preparative purification was dissolved in phosphate buffer (0.2M, pH 7.4) to give a final concentration of 40 mg / mL. Further, this solution was diluted 10-fold with phosphate buffered saline (PBS, pH 7.4) to a final concentration of 4 mg / mL. The carrier protein solution was prepared as follows. A lyophilized powder of mouse serum albumin (MSA, Sigma-Aldrich) was dissolved in PBS to a final concentration of 10 mg / mL. In 1.5 mL of Eppendorf Safe-Lock Tubes, 4 mg / mL GAL13 solution (500 μL) and 10 mg / mL MSA solution (200 μL) were mixed. Subsequently, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo Scientific) was dissolved in ultrapure water to prepare a 10 mg / mL aqueous solution. A 10 mg / mLEDC aqueous solution (100 μL) was quickly added to a mixed solution of GAL13 and MSA (700 μL), mixed by inversion, and then incubated overnight at room temperature.

Immediately after completion of the reaction, the reaction solution was gel-filtered with Zeba Spin Desalting Columns (7K MWCO, Thermo Scientific). Furthermore, using a Slide-A-Lyzer MINI Dialysis Device (3.5K MWCO, Thermo Scientific), dialysis was performed at a low temperature (4 ° C.). The external dialysis solution was PBS. Three hours after the start of dialysis, the external fluid was exchanged, and dialysis was further performed overnight. After completion of dialysis, the solution in the dialysis device was collected and concentrated with Amicon Ultra-0.5 (30K MWCO, Merck). The protein concentration of MSA (GAL13-MSA) cross-linked with GAL13 was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific), and adjusted to an arbitrary protein concentration with PBS. GAL13-MSA was used as an immune antigen in the production of mouse monoclonal antibodies.

In the Enzyme-Linked ImmunoSorbent Assay (ELISA), GAL13 was crosslinked with bovine serum albumin (BSA, Sigma-Aldrich) and used in the test as GAL13-BSA. In addition, as a negative control group, N ^α , N ^ε -dicarbobenzoxy-L-lysine (Z-Lys (Z) -OH, Watanabe Kagaku Kogyo) was crosslinked with BSA to prepare Z-Lys-BSA. .. These samples were filtered and sterilized (pore size 0.22 μm) in a clean bench as needed.

[Example 3] Preparation of monoclonal antibody (1) Immunity to mice As an immune antigen, a novel structure modification derived from Glycer-AGEs-mouse serum albumin (GAL13-MSA, 1 mg / mL) was added to Freund's Adjuvant, Complete (1 mg / mL) in the same volume. An emulsion was prepared by mixing with Sigma-Aldrich (antigen concentration: 0.5 mg / mL), and the back subcutaneous of 5 BALB / c mice was first immunized (antigen amount: 200 μg / animal). Boost immunization (antigen) every other week using an emulsion (antigen concentration: 0.5 mg / mL) prepared by mixing GAL13-MSA (1 mg / mL) with the same volume of Freund's Adjuvant, Incomplete (Sigma-Aldrich). Amount: 50 μg / animal). After immunization 6 times from the initial immunization, blood was collected from the tail vein to confirm the antibody titer. An emulsion for booster immunization (amount of antigen: 50 μg) was finally administered intraperitoneally to the mouse having a high antibody titer, and 3 days later, the spleen was removed for cell fusion.

(2) Measurement of antibody titer The titer of antiserum was evaluated by ELISA. In a 96-well microtiter plate (NUNC), in the immune antigen GAL13-MSA, a novel structure modification derived from Glycer-AGEs in which the carrier protein was changed to BSA-bovine serum albumin (GAL13-BSA) was added at a concentration of 1 μg / mL. 50 μL / well was added and solidified at 4 ° C. overnight. After washing 3 times with PBS (PBS-T) containing 0.05% (v / v) Tween 20, blocking was performed with a carbonate buffer (pH 9.5) containing 0.5% (w / v) gelatin for 1 hour. The antiserum was serially diluted 1000 to 3 times, and after preparing a dilution series up to 729000 times, 50 μL / well was placed on an antigen-immobilized plate and allowed to stand for 1 hour. After washing, 50 μL / well of secondary antibody (Horseradish peroxidase (HRP) -labeled anti-mouse IgG (ZYMED)) diluted 2500-fold with PBS-T was added and allowed to stand for 1 hour. After washing, 0 in each well. An o-phenylenediamine solution (100 μL) prepared to 0.5 mg / mL with 0.1 M phosphate buffer (pH 5.0) containing 0.02% (w / v) hydrogen peroxide was added, and 25 After allowing to stand at ° C. for 10 minutes, a 1 M sulfuric acid solution (100 μL) was added to each well to stop the coloring reaction. Then, the absorbance at 490 nm was measured with a microplate reader. As a result, 9000 times or more of the anti-serum. Mice that showed significant reactivity with the antigen in the diluted solution of were used for cell fusion.

(3) Preparation of spleen cells and cell fusion The spleen extracted from the mouse was ground to prepare ^{about 1 × 10 8 spleen cells per mouse.} P3U1 which is a myeloma cell was cultured, and P3U1 having a viable cell rate of 95% or more was prepared on the day of cell fusion. The spleen cells and P3U1 were mixed at a ratio of 5: 1 (cell number ratio), and cell fusion was performed with polyethylene glycol having a molecular weight of 1,450 at a concentration of 50% (w / v). After the fusion, the cells were washed with a medium, suspended in a HAT medium, ^{and the cells were seeded into each well of a 96-well culture plate so as to be 1 × 10 5} cells / well, and a hybridoma was selectively cultured. On the 10th day of cell fusion, the hybridoma culture supernatant was collected, and the antibody titer in the culture supernatant was measured.

(4) Screening of antibody production positive wells The culture supernatant on the 10th day after cell fusion was collected, and antibody production positive wells were screened by the above antibody titer measurement method. Clone was selected for GAL13-BSA, glyceraldehyde-derived AGEs-containing BSA (Glycer-AGEs-BSA) positive, and Z-lysine-modified bovine serum albumin (Z-Lys-BSA) negative.

(5) Cloning A clone having high specificity for GAL13-BSA was cloned by the limiting dilution method. That is, cells were prepared at 5 cells / mL in RPMI medium containing 10% FCS, and 200 μL was added to each well of two 96-well culture plates. After 10 days, it was confirmed that the culture supernatant was positive for GAL13-BSA, Glycer-AGEs-BSA, and negative for Z-Lys-BSA, and clones derived from each well were obtained. As an antibody of the present invention having sufficient specificity, a novel monoclonal antibody SJ-5 producing cell was obtained.

(6) Purification of antibody The cells producing the novel monoclonal antibody SJ-5 were cultured in RPMI medium containing 10% FCS, washed with PBS, and in serum-free medium (SMF medium, Thermo Scientific) for 4 days to 6 days. The cells were cultured for one day to obtain a culture supernatant. The culture supernatant was purified from the IgG fraction on a Protein G column (manufactured by GE Healthcare) to obtain a novel monoclonal antibody SJ-5 as an antibody of the present invention having sufficient specificity.

[Example 4] Peroxidase (POD) labeling method for monoclonal antibody and polyclonal antibody The obtained novel monoclonal antibody SJ-5 (PBS solution, 200 μg) or anti-glyceraldehyde-derived AGEs polyclonal antibody (Molecular Medicine 6 (2): The PBS solution (200 μg) according to Takeuchi et al.'S method described in 114-125, 2000 was POD-labeled using the Peroxidase Labeling Kit-SH (Dojin Chemical Laboratory). The labeling method followed the procedure in the instruction manual. The obtained POD-labeled antibody solution was mixed with glycerol (Sigma-Aldrich) at a ratio of 1: 1 and stored at −20 ° C.

[Example 5] Confirmation of reactivity of novel monoclonal antibody by ELISA direct method Glycer-AGEs-derived novel structure modification-BSA (GAL13-BSA) was added at a concentration of 1 μg / mL at a concentration of 100 μL / well, and the temperature was 4 ° C. overnight. It was immobilized with. After washing 3 times with PBS (PBS-T) containing 0.05% (v / v) Tween 20, blocking was performed with PBS-T containing 0.5% (w / v) gelatin for 1 hour. The POD-labeled novel monoclonal antibody SJ-5 prepared in Example 4 and the anti-glyceraldehyde-derived AGEs polyclonal antibody as a control were serially diluted 4-fold from 4 μg / mL to prepare a dilution series up to 0.06254 μg / mL. Then, 100 μL / well was placed in the antigen-immobilized plate and allowed to stand for 1 hour. After washing, an o-phenylenediamine solution (o-phenylenediamine solution) prepared to 0.5 mg / mL with 0.1 M phosphate buffer (pH 5.0) containing 0.02% (w / v) hydrogen hydrogen in each well. 100 μL) was added and allowed to stand at 25 ° C. for 10 minutes, and then a 1 M sulfuric acid solution (50 μL) was added to each well to stop the color reaction. The absorbance at 490 nm was then measured with a microplate reader.

As a result, as shown in FIG. 1, it was shown that the POD-labeled SJ-5 antibody responded well to GAL13-BSA.

[Example 6] Confirmation of reactivity of novel monoclonal antibody by ELISA competition method GAL13-BSA was added at a concentration of 1 μg / mL at a concentration of 100 μL / well, and immobilized at 4 ° C. overnight. After washing 3 times with PBS (PBS-T) containing 0.05% (v / v) Tween 20, blocking was performed with a carbonate buffer (pH 9.5) containing 0.5% (w / v) gelatin for 1 hour. After preparing POD-labeled SJ-5 antibody (0.4 μg / mL) and anti-glyceraldehyde-derived AGEs polyclonal antibody (6 μg / mL) as a control, GAL13-BSA and 10 of BSA as a control were placed on an antigen-immobilized plate. A double dilution series solution (50 μL each) and POD-labeled SJ-5 antibody or anti-glyceraldehyde-derived AGEs polyclonal antibody (50 μL) were added as a control, and the mixture was stirred with a plate mixer and allowed to stand at room temperature for 1 hour. After washing, 100 μL of o-phenylenediamine solution prepared to 0.5 mg / mL with 0.1 M phosphate buffer (pH 5.0) containing 0.02% (w / v) hydrogen hydrogen in each well. Was added and allowed to stand at 25 ° C. for 10 minutes, and then 50 μL of 1 M sulfuric acid solution was added to each well to stop the color reaction. The absorbance at 490 nm was then measured with a microplate reader. FIG. 2 shows the results of the anti-glyceraldehyde-derived AGEs polyclonal antibody, and FIG. 3 shows the results of the POD-labeled SJ-5 antibody.

Both POD-labeled SJ-5 antibody and anti-glyceraldehyde-derived AGEs polyclonal antibody were shown to react well with GAL13-BSA even in the competitive method, and the antibody concentration used for POD-labeled SJ-5 antibody was anti-glyceride. It was shown that similar results were obtained at a concentration 15 times lower than that of glyceraldehyde-derived AGEs polyclonal antibody.

[Example 7] Confirmation of specificity of novel monoclonal antibody by ELISA competition method GAL13- prepared in 1 μg / mL of PBS (pH 7.4) as an immobilized antigen in each well of a 96-well microtiter plate (NUNC). 100 μL of BSA or Glycer-AGEs-BSA was added, and the mixture was left at 25 ° C. for 1 hour. It was solid-phased at 4 ° C. overnight. After washing 3 times with PBS (PBS-T) containing 0.05% (v / v) Tween 20, blocking was performed with a carbonate buffer (pH 9.5) containing 0.5% (w / v) gelatin for 1 hour. POD-labeled SJ-5 antibody (0.4 μg / mL) was prepared under the same conditions as in Example 6, and glucose-derived AGEs-containing BSA (Glu-AGEs-BSA) was placed on an antigen-immobilized plate to confirm specificity. glycolaldehyde derived AGEs containing BSA (glycol-AGEs-BSA) , methylglyoxal-derived AGEs containing BSA (MGO-AGEs-BSA) , glyoxal derived AGEs containing BSA (GO-AGEs-BSA) , N ε - carboxymethyl lysine-containing BSA ( CML-BSA), N ^ε -carboxyethyl lysine-containing BSA (CEL-BSA), and bovine serum albumin (BSA) were similarly added in an amount of 50 μL each, and then POD-labeled SJ-5 antibody (50 μL) was added, and the plate was added. After stirring with a mixer, the mixture was allowed to stand at room temperature for 1 hour. After washing, an o-phenylenediamine solution prepared at 0.5 mg / mL with 0.1 M phosphate buffer (pH 5.0) containing 0.02% (w / v) hydrogen hydrogen in each well ( 100 μL) was added and allowed to stand at 25 ° C. for 10 minutes, and then 50 μL of 1 M sulfuric acid solution was added to each well to stop the color reaction. The absorbance at 490 nm was then measured with a microplate reader.

FIG. 4 shows the specificity of the POD-labeled SJ-5 antibody when GAL13-BSA was used as the immobilized antigen, and FIG. 5 shows POD labeling when Glycer-AGEs-BSA was used as the immobilized antigen. As a result of the specificity of the novel monoclonal antibody SJ-5, the absorbance when the control BSA is 1 is shown.
Even when GAL13-BSA and Glycer-AGEs-BSA were immobilized, the specificity of the novel monoclonal antibody SJ-5 was positive for Glycer-AGEs-BSA and Glycol-AGEs-BSA, and Glu-AGEs-BSA and Fru. It was negative for -AGEs-BSA, MGO-AGEs-BSA, GO-AGEs-BSA, CML-BSA, and CEL-BSA.

[Example 8] Measurement of dissociation constant of monoclonal antibody The dissociation constant (K _d value) of the monoclonal antibody SJ-5 was measured by the following method. Glyceraldehyde-derived AGEs-containing BSA (Glycer-AGEs-BSA) was diluted with a 10 mM sodium acetate solution (pH 4.0) to prepare a ligand solution having a final concentration of 100 μg / mL. Biacore T200 (GE Healthcare) was used for ligand immobilization and KD value calculation. The ligand solution was immobilized on the sensor chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare). Subsequently, an antibody diluent prepared by diluting the monoclonal antibody to a concentration of 0 to 400 nM was prepared, and the K _d value was calculated.

As a result, the K _d value of the monoclonal antibody was 57.4 nM. Further, BSA (GAL13-BSA) in which the peaks separated by HPLC were chemically bonded was diluted with a 10 mM sodium acetate solution (pH 5.0) to prepare a ligand solution having a final concentration of 25 μg / mL. _{As a result of calculating the Kd} value of the monoclonal antibody in the same manner as above, it was 87.5 nM.

[Example 9] Mass spectrometry of GAL13 The solvent used in the mass spectrometry was HPLC grade of Fujifilm Wako Pure Chemical Industries, Ltd. The dry powder of GAL13 prepared in Example 2 was dissolved in a 0.1% TFA solution containing 50% acetonitrile to give a final concentration of 0.25 mg / mL. For the matrix, α-cyano-4-hydroxysilicic acid (CHCA) was purchased from Shimadzu LLC, and a 10 mg / mL solution was prepared with a 0.1% TFA solution containing 50% acetonitrile. The sample solution and the matrix solution were mixed in equal amounts in 0.6 mL of Eppendorf Safe-Lock Tubes, and then 1 μL was added dropwise to the wells of the measuring plate and air-dried. As a mass calibration peptide, ^{a mixed solution of des-Arg 1} -Bradykinin, Angiotensin I, Glu ¹ -Fibrinopeptide (AB SCIEX) and a matrix was prepared and dropped onto a plate in the same manner. The plate after air drying was inserted into AXIMA Performance (Shimadzu Corporation), which is a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF-MS), and cation measurements were performed in the reflector mode. The result of mass spectrometry is shown in FIG.

As a result, sample-derived peaks were observed at m / z = 650-750 and 953. In particular, m / z = 691.2926 is _{a Na adduct of C 34} H ₄₄ N ₄ O ₁₀ [M + Na] ⁺ ion (theoretical value of monoisotopic mass 691.2957, error 4.5 ppm from the measured value). ) Was considered. This is approximately the same as the monoisotopic mass of the structure in which the Z-Lys-OH2 molecule is crosslinked by the DL-GLA2 molecule. Further, m / z = 953.3945 is _{a Na adduct of C 48} H ₆₂ N ₆ O ₁₃ , [M + Na] ⁺ ion (theoretical value of monoisotopic mass 953.4276, error 34.7 ppm from the measured value). ) Was considered. This is approximately the same as the monoisotopic mass of the structure in which the Z-Lys-OH3 molecule is crosslinked by the DL-GLA2 molecule. From the above results, it was shown that GAL13 contains at least the following two kinds of compounds.

[Example 10] Measurement of peak radicals separated by HPLC The dry powder of GAL13 prepared in Example 2 was dissolved in 0.2 M phosphate buffer (pH 7.4) to a final concentration of 50 mg / mL. 50 μL of this solution was collected with a graduated glass capillary micropipette (Drummond Scientific Company), and the lower end of the capillary was sealed with an EM Meister hematocrit capillary sealing wax plate (As One). Then, the capillary tube was transferred to a standard sample tube for an electron spin resonance (ESR) device (outer diameter 4 mm, Shigemi Co., Ltd.) and inserted into an ESR resonator. An ELEXSYS-II E580 (Bruker) was used as the ESR device, and the room temperature was measured by the continuous wave method. The measurement conditions are as follows:
Field center 3350G;
Field width 150G ；
Averaged scan 20;
Sampling time 0.03s ；
Field modulation amplitude 0.4mT;
Field modulation Frequency 100kHz;
Microwave power 3mW;
Receiver gain 60.

The results are shown in FIG. From the g value (2.443) and the linearity of the spectrum, it was found that the peaks fractionated by HPLC were AGEs derived from glyceraldehyde containing carbon centers that generate radicals.

[Example 11] Evaluation of oxidative activity of GAL13 (1) Preparation of 3,3'-diaminobenzidine (DAB) DAB was purchased from Dojin Chemical Research Institute. DAB was weighed and dissolved in Tris buffer (50 mM, pH 7.4) to a final concentration of 5 mg / mL.

(2) Preparation of sample solution The dry powder of GAL13 prepared according to Example 2 was dissolved in a phosphate buffer solution (0.2 M, pH 7.4) to prepare a 2 mg / mL solution. A 2 mg / mL Z-Lys-OH solution was also prepared as a negative control group.

(3) Evaluation Method of Oxidative Activity 10 μL of 5 mg / mL DAB solution or Tris buffer (50 mM, pH 7.4) was added to a 96-well plate (Violamo). Then, 90 μL of the sample solution was added, and the mixture was stirred with a plate mixer. After incubating the plate at 37 ° C. for 3 hours under shading, the absorbance at 460 nm was measured with a Cytion5 plate reader (BioTek). The oxidative activity against DAB was determined by subtracting the absorbance at 460 nm in the Tris buffer addition group from the absorbance at 460 nm in the DAB-added group. The results are shown in the graph of FIG. The average value and standard deviation of the three measured values are shown. For statistical analysis, Student's t-test was performed by JMP14.0 (SAS), and it was found that the oxidizing activity of GAL13 against DAB was significantly higher than that of Z-Lys-OH (P <0.01).

[Example 12] Preparation of glyceraldehyde-derived pyridinium compound (GLAP) GLAP can be prepared by a known method (Usui et al., Biosci. Biotechnol. Biochem., 2003, 67 (4), 930-932. ). Specifically, GLAP was prepared by the following method.

(1) Preparation of GLAP reaction solution N ^alpha - acetyl -L- lysine (Ac-Lys-OH, Tokyo Kasei Kogyo), DL-glyceraldehyde (DL-GLA, Nacalai Tesque) was obtained by purchase.

DL-GLA (360.3 mg) was weighed and transferred to a 50 mL centrifuge tube. Phosphate buffer (0.2 M, pH 7.4, 20 mL) was added to the tube, and the mixture was stirred with a vortex mixer until DL-GLA was dissolved. Then, Ac-Lys-OH (376.5 mg) was added and stirred until dissolved (DL-GLA concentration in solution: 200 mM, Ac-Lys-OH concentration: 100 mM). The tube lid was sealed with a film such as Parafilm ™ to prevent the liquid in the tube from evaporating, and the tube was incubated at 37 ° C. for 1 week. The obtained reaction solution was stored at 4 ° C.

(2) Separation and Purification of GLAP The GLAP reaction solution was filtered through a syringe filter (Millex-LG, Merck) having a pore size of 0.2 μm, and the filtrate was used as a sample of a liquid chromatograph mass spectrometer (LC-MS). The LC section of the LC / MS consisted of a 1260 Infinity II system (Agilent Technology), and the MS section consisted of a quadrupole mass spectrometer (InfinityLab LC / MSD, G6125B, Agilent Technologies). The LC unit consists of the following modules; quarterly pump (G7111B), isocratic pump (G7110B), multi-sampler (G7167A), multi-column thermostat (G7116A), diode array detector (G7115A), fraction. Collector (G1364F), MS flow modulator (G7170B), OpenLAB CDS Pump Station software. The mobile phase was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. in HPLC grade. The mobile phase A was a 10 mM ammonium acetate aqueous solution obtained by mixing 10 mL of a 1 M ammonium acetate solution and 990 mL of distilled water, and the mobile phase B was acetonitrile. The analysis time was 0-7 minutes with mobile phase A: B = 99: 1, and 7.1-12 minutes with mobile phase A: B = 10: 90. The column used was ZORBAX SB-C18 (150 x 9.4 mm, Agilent Technologies) and the flow rate was set to 4 mL / min. 80 μL of the filtered sample solution was injected into the LC-MS apparatus, and the peak of GLAP was detected by a diode array detector (215 nm and 254 nm) and MS (cation detection). At the analysis time of 5 minutes, a peak having UV absorption was observed, and the peak contained a cation of m / z = 297.1. Therefore, by performing MS preparative with m / z = 297 as a trigger, the peak with a retention time of 5 minutes was recovered. The solution after separation was concentrated to dryness with a rotary evaporator (Tokyo Rika Kikai). The resulting precipitate was stored at 4 ° C.

(3) Confirmation of GLAP structure The obtained precipitate (8 mg) is dissolved in heavy water (0.6 mL, Fujifilm Wako Pure Chemical Industries, Ltd.) and transferred to a sample tube (Shigemi) for nuclear magnetic resonance (NMR) with an outer diameter of 5 mm. bottom. After that, the structure of GLAP was confirmed with an NMR device (AVANCE III HD, 500 MHz, CryoProbe mounted type, Bruker). The attribution of total hydrogen and carbon was determined from ¹ H-NMR and ^{13 C-NMR spectrum data. Further, 1 H- 1 H COSY, 1} H- 13 C HSQC, 1 H- 13 C HMBC, 1 H- 15 N HSQC, 1 H- 15 N HMBC spectra by analyzing the confirmed the validity of the assignment .. The structure of GLAP is shown below.

[Example 13] Reactivity evaluation of novel monoclonal antibody and GLAP by competing ELISA (1) Reagent preparation The following reagent was dissolved in RO water to make 1 L, and used as a coating solution.
Sodium carbonate (Fujifilm Wako Pure Chemical Industries) 1.59g
Sodium hydrogen carbonate (Fujifilm Wako Pure Chemical Industries, Ltd.) 2.93 g
BSA (5 g, Sigma-Aldrich) was dissolved in PBS (500 mL) and used as a blocking solution.

50 mM Tris (6.1 g, Wako Pure Chemical Industries, Ltd.) was dissolved in RO water (about 900 mL), and the pH was adjusted to 7.4 with 6N hydrochloric acid (Wako Pure Chemical Industries, Ltd.). Glycerol (1 mL, Sigma-Aldrich) and Tween 20 (1 mL, Nacalai Tesque) were added to the obtained solution, and the volume was increased to 1 L with RO water. The resulting solution was used as a diluent.

The following reagents were dissolved in RO water to make 1 L, and 9 L of RO water was further added, and then Tween 20 (5 mL) was added. The obtained solution was used as a washing solution.
Sodium chloride (Fujifilm Wako Pure Chemical Industries) 80g
Potassium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) 2g
Disodium hydrogen phosphate dodecahydrate (Fujifilm Wako Pure Chemical Industries, Ltd.) 29 g

(3) Immobilization of antigen A solution of glyceraldehyde-derived AGEs-containing BSA (stock solution: 10 mg / mL PBS solution) prepared at 1 μg / mL in the coating solution was added to a 96-well microtiter plate (COSTAR) of 100 μL each. Incubated overnight at 4 ° C.

(4) Blocking Each well treated for solid phase was washed 3 times with a washing solution (300 μL), a blocking solution (200 μL) was added, and the mixture was left at room temperature for 1 hour.

(5) Competitive Experiment GAL13 (prepared in Example 2), GLAP (prepared in Example 12), and Z-Lys-OH (Tokyo Chemical Industry) were added to phosphate buffer (0.2M, pH 7.4), respectively. To prepare a 20 mg / mL solution. Further, by diluting with a diluted solution, a sample solution of a 2-fold dilution series (6 points) of 0.0063 to 2 mg / mL was prepared. A POD-labeled novel monoclonal antibody (SJ-5) solution was diluted 16,000 times with a diluted solution containing BSA (1 mg / mL, Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a POD-labeled SJ-5 antibody diluted solution.

Each well treated with the blocking solution was washed 3 times with a wash solution (300 μL), a 2-fold dilution series of sample solution (50 μL each) and a POD-labeled SJ-5 antibody dilution solution (50 μL) were added, and 2 with a plate mixer. After stirring for 1 minute, the mixture was incubated at 25 ° C. for 1 hour.

(6) Color development After washing 3 times with a washing solution (300 μL), 100 μL of a substrate solution (ELISA POD substrate TMB kit (Popular), Nacalai Tesque) was added, and the mixture was incubated at room temperature for 10 minutes under shading. Then, 2N sulfuric acid (50 μL) was added to stop the color development.

(7) Absorbance measurement and data analysis The absorbance at the main wavelength of 450 nm and the sub-wavelength of 650 nm was measured with a microplate reader (Cytion5, BioTek), and the absorbance of the sub-wavelength was subtracted from the absorbance of the main wavelength. The result is shown in FIG. In GAL13, the absorbance decreased in a concentration-dependent manner, but in GLAP and Z-Lys-OH, no decrease in absorbance was observed. Therefore, it was revealed that the novel monoclonal antibody SJ-5 is an antibody that does not recognize GLAP, which is a known glyceraldehyde-derived AGEs.

[Example 14] Neutralization test for suppressing vascular endothelial cell lumen formation by Glycer-AGEs-BSA using SJ-5 (1) Preparation of Matrigel matrix The Matrigel matrix (Corning) was placed in a refrigerator and thawed overnight. .. The thawed Matrigel matrix was added to a 12-well plate (Corning) at 300 μL / well and _{allowed to stand at 37 ° C. in a CO 2} incubator (ESPEC) for 1 hour to cure.

(2) Preparation of antibody reaction solution with Glycer-AGEs-BSA or control BSA 10 μL of Glycer-AGEs-BSA (10 mg / mL) or 10 μL of control BSA (10 mg / mL) prepared in Example 1 and phosphate buffered saline (PBS, Wako), SJ-5 (10 mg / mL) or 90 μL of control antibody (10 mg / mL) was added to a 1.5 mL tube (Watson) and allowed to stand at room temperature for 10 minutes. Then, it was centrifuged at 14000 rpm for 15 minutes using a centrifuge (Thermo Fisher Scientific), and the supernatant was collected.

(3) Cavity formation test HUVEC (human umbilical vein vascular endothelial cells, National Institutes of Biomedical Innovation, Health and Nutrition JCRB Cell Bank) is a 10 cm dish (corning) and 10 mL of HUVEC medium (KAC). Was cultured using.

The cultured HUVEC was washed with 10 mL of PBS, added with 1 mL of trypsin-EDTA (Wako), and allowed to stand at room temperature for 3 minutes. After standing, it was confirmed that HUVEC had peeled off from the dish, and trypsin-EDTA was neutralized by adding 10 mL of HUVEC medium. After neutralization, the whole volume was transferred to a 50 mL tube (Corning) and centrifuged at 1500 rpm for 3 minutes using a centrifuge (Kubota).

After centrifugation, the supernatant was discarded, 1 mL of HUVEC medium was added to the precipitated cells, and the cells were resuspended. 10 μL of the suspension was taken, mixed with 10 μL of trypan blue (NanoEnTek), and 10 μL was added to the hemocytometer (NanoEnTek). Then, the number of viable cells was counted by the auto cell counter EVE (NanoEnTek). So that the cell suspension 2.5x10 ⁵ cells / mL diluted in HUVEC medium, and plated in 400 [mu] L / well Matrigel prepared in (1). After sowing, a solution prepared in (2) adjusted to 100 μg / mL with PBS was added at 100 μL / well, and the mixture was _{cultured in a CO 2} incubator for 8 hours.

FIG. 13 shows the morphology of HUVEC when observed in a bright field using a 20x objective lens of BZ-X710 (Keyence) after culturing for 8 hours. As shown in the figure, in the group to which the control BSA was added, HUVEC was confirmed to form lumens in the SJ-5 antibody and the control antibody reaction solution Matrigel matrix. However, Glycer-AGEs-BSA inhibited lumen formation. In addition, this inhibition of lumen formation was neutralized with the reaction solution with the SJ-5 antibody, but the neutralization could not be confirmed with the control antibody. From this result, it was clarified that SJ-5 can neutralize the effect of Glycer-AGEs-BSA on HUVEC.

[Example 15] Endothelial mesenchymal conversion inhibition test by Glycer-AGEs-BSA using SJ-5 antibody (1) The Matrigel matrix was prepared in the same manner as in Example 14.

(2) Preparation of antibody reaction solution with Glycer-AGEs-BSA or control BSA 10 μL of Glycer-AGEs-BSA (10 mg / mL) prepared in Example 1 and PBS, SJ-5 (10 mg / mL) or control antibody (10 mg) / ML) 90 μL was added to a 1.5 mL tube (Watson). In addition, 90 μL of PBS solution of control BSA (10 mg / mL) was added. These solutions were allowed to stand at room temperature for 10 minutes. Then, it was centrifuged at 14000 rpm for 15 minutes using a centrifuge (Thermo Fisher Scientific), and the supernatant was collected.

(3) Lumen formation inhibition test HUVEC was cultured in a 10 cm dish using 10 mL of HUVEC medium.
The cultured HUVEC was washed with 10 mL of PBS, 1 mL of trypsin-EDTA was added, and the cells were allowed to stand at room temperature for 3 minutes. After standing, it was confirmed that HUVEC had peeled off from the dish, and trypsin-EDTA was neutralized by adding 10 mL of HUVEC medium. After neutralization, the whole volume was transferred to a 50 mL tube and centrifuged at 1500 rpm for 3 minutes using a centrifuge.

After centrifugation, the supernatant was discarded, 1 mL of HUVEC medium was added to the precipitated cells, and the cells were resuspended. 10 μL of the suspension was taken, mixed with 10 μL of trypan blue, and 10 μL was added to the hemocytometer. Then, the number of living cells was counted by the auto cell counter EVE. So that the cell suspension 2.5x10 ⁵ cells / mL diluted in HUVEC medium, and plated in 400 [mu] L / well Matrigel prepared in (1). After sowing, each reaction solution prepared in (2) was added at 100 μL / well and _{cultured in a CO 2} incubator for 8 hours.

(4) Cell Recovery Each well was washed 3 times with 1 mL PBS and a cell recovery solution (Corning) was added at 500 μL / well.
Cells and Matrigel matrix were scraped with a blue chip (Watson), collected in 1.5 mL tubes, wells rerinsed with a 500 μL cell recovery solution and transferred to 1.5 mL tubes. After mixing the 1.5 mL tube 5 times, it was allowed to stand on ice for 1 hour, and it was confirmed that the Matrigel matrix was completely decomposed. After decomposing the Matrigel matrix, centrifugation was performed at 4 ° C. and 800 rpm, and the supernatant was discarded. Gently suspend the precipitated cells in PBS with 1 mL of ice-cooled, centrifuge at 4 ° C. and 800 rpm, discard the supernatant, and gently suspend the reprecipitated cells in PBS with 1 mL of ice-cooled 4 Centrifugation was performed at 800 ° C. and 800 rpm.

(5) mRNA recovery mRNA recovery was ^{performed using the CellAmp TM} Direct RNA Prep Kit for RT-PCR (Takara). The actual operation is shown below.
Centrifugation was performed, and 125 μL of Cell Amp Washing Buffer attached to Kit was added to the precipitated cells and mixed. Centrifugation was performed at 300 xg for 5 minutes, the supernatant was removed, and 49 μL of Cell Amp Processing Buffer attached to Kit and 1 μL of DNase I for Direct RNA Prep were added to each tube. After allowing to stand at room temperature for 5 minutes, the mRNA was transferred to a PCR tube (Nippon Genetics) and allowed to stand at 75 ° C. for 5 minutes using a thermal cycler (Nippon Genetics) to obtain mRNA.

(6) Reverse transcription reaction The reverse transcription reaction was ^{carried out using PrimeScript TM} RT Master Mix (Takara). The actual operation is shown below. 1 μL of mRNA obtained in (5), 5 × 2 μL of PrimeScript RT Master Mix and 7 μL of RNase Free dH ₂ O attached to Master Mix were mixed with a PCR tube and reacted at 37 ° C. for 15 minutes and 85 ° C. for 5 seconds using a thermal cycler. ..

Ｐｒｉｍｅｒは下記の配列を株式会社ファスマックに合成委託して入手したものを使用した。
ヒトα−ＳＭＡα−ＳＭＡ
フォワード：ＣＧＧＣＴＴＴＧＣＴＧＧＧＧＡＣＧＡＴ
リバーズ：ＣＡＧＧＧＧＣＡＡＣＡＣＧＡＡＧＣＴＣＡＴ
ヒトＣＤ３１
フォワード：ＡＴＴＧＣＡＧＴＧＧＴＴＡＴＣＡＴＣＧＧＡＧＴＧ
リバーズ：ＣＴＣＧＴＴＧＴＴＧＧＡＧＴＴＣＡＧＡＡＧＴＧＧ
ヒトβ−アクチン
フォワード：ＣＴＧＧＡＡＣＧＧＴＧＡＡＧＧＴＧＡＣＡ
リバーズ：ＡＡＧＧＧＡＣＴＴＣＣＴＧＴＡＡＣＡＡＴＧＣＡ (7) Real-time PCR
Real-time PCR was performed using Luna Universal qPCR Master Mix (BioLabs). The reverse transcription reaction product obtained in (6) was diluted with 90 μL of ultrapure water (Thermo Fisher Scientific). After dilution, a reaction solution was prepared according to the composition shown in the table below, and added to a 384-well plate (Nippon Genetics).

Primer used the following sequence obtained by entrusting synthesis to Fasmac Co., Ltd.
Human α-SMA α-SMA
Forward: CGGCTTTGGCTGGGGAGAGAT
Rivers: CAGGGGCAACACGAAGCTCAT
Human CD31
Forward: ATTGCAGTGGTTATCATCGGAGTG
Rivers: CTCGTTGTTGGAGTTCAGAGTGG
Human β-actin forward: CTGGAACGGGTGAAGGTGACA
Rivers: AAGGGACTTCCTGTAACATGCA

After the addition ^{, real-time PCR was performed with a Quant Studio TM} 12K Flex real-time PCR system (Thermo Fisher Scientific) to quantify the gene expression level.

CD31 is known as a marker for endothelial cells, and α-smooth muscle actin (α-SMA) is known as a marker for mesenchymal cells (Non-Patent Document 14, Figure 2, etc.). The graphs in which the expression levels of α-SMA and CD31 were evaluated by calculating the expression levels of β-actin as an internal standard are shown in FIGS. 14 and 15. From FIG. 14, it was clarified that the expression level of α-SMA was increased by the addition of Glycer-AGEs-BSA. In addition, this effect can be suppressed by SJ-5, but not by the control antibody. Further, from FIG. 15, it was clarified that the expression level of CD31 was decreased by the addition of Glycer-AGEs-BSA for CD31. In addition, this effect can be suppressed by SJ-5, but not by the control antibody. The increase in the expression level of α-SMA and the decrease in the expression level of CD31 are the effects observed in the endothelial mesenchymal transformation in which vascular endothelial cells are transformed into mesenchymal cells. From these results, it was confirmed that Glycer-AGEs-BSA has an action of inducing endothelial mesenchymal conversion, and SJ-5 can suppress this action.

[Example 16] Preparative purification (purification) of Glycer-AGEs-Z-Lys
Glycer-AGEs-Z-Lys obtained as a precipitate in Example 2 was dissolved in 0.2 M phosphate buffer (pH 7.4) to prepare a sample solution of 50 mg / mL. Then, the mixture was filtered through a syringe filter (Millex-LG, Merck) having a pore size of 0.2 μm, and the filtrate was used as a sample of a liquid chromatograph mass spectrometer (LC / MS). The LC unit and the MS unit of the LC / MS were configured in the same manner as in Example 12. The mobile phase was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. in HPLC grade. The mobile phase A was a 10 mM ammonium acetate aqueous solution obtained by mixing 10 mL of a 1 M ammonium acetate solution and 990 mL of distilled water, and the mobile phase B was acetonitrile. At the analysis time of 0-2 minutes, the cells flowed down with a concentration gradient of mobile phase A: B = 100: 0-85: 15. At an analysis time of 2-15 minutes, the concentration gradient of mobile phase A: B = 85: 15-70: 30, and at 15-17 minutes, mobile phase A: B = 70: 30-10: 90 flowed down. The column used was ZORBAX SB-C18 (150 x 9.4 mm, Agilent Technologies) and the flow rate was set to 4 mL / min. 50 μL of the filtered sample solution was injected into the LC / MS apparatus, and the peak of Glycer-AGEs-Z-Lys was detected by a diode array detector (260 nm) and MS (cation detection). At an analysis time of 12.3 minutes, a UV-absorbing peak was observed, which contained ^{[M + Na] + at m / z = 691.3.} Therefore, the peak with a retention time of 12.3 minutes was recovered by performing MS preparative with m / z = 691 as a trigger. Hereinafter, this peak will be referred to as GAL691. The LC / MS analysis results are shown in FIG. 16 (diode array detection (260 nm)), FIG. 17 (UV spectrum), and FIG. 18 (MS spectrum).

Acetonitrile contained in the preparative solution was removed by a rotary evaporator (Tokyo Rika Kikai). To 5 mL of the residue solution, 1 mL of a 10% TFA aqueous solution was added and mixed by inversion. Then, it was centrifuged (12,000 × g) for 10 minutes at room temperature, and the supernatant was removed. Ultrapure water (5 mL) was added to the obtained precipitate, and centrifugation was performed again to remove the supernatant. The precipitate was washed by performing this operation a total of three times. The precipitate after washing was air-dried, the dry weight was measured (1 mg), and the precipitate was stored in a refrigerator.

[Example 17] Preparation of glyceraldehyde-derived pyridinium compound (Lys-hydroxy-triocidin) Lys-hydroxy-triocidin can be prepared by a known method (Non-Patent Document 12). Specifically, Lys-hydroxy-triocidin was prepared by the following method.

(1) Preparation of Lys-hydroxy-triocidin reaction solution Methanol (Fujifilm Wako Pure Chemical Industries, Ltd.), diethylenetriamine-N, N, N', N ", N" -pentaacetic acid (DTPA, Dojin Chemical Research Institute), N ^α- Acetyl-L-lysine (Ac-Lys-OH, Tokyo Kasei Kogyo) and DL-glyceraldehyde (DL-GLA, Nacalai Tesque) were obtained by purchase.
Phosphate buffer (0.267M, pH 10.5, 30 mL) and methanol (10 mL) were mixed in a 50 mL centrifuge tube (0.2 M phosphate buffer containing 25% methanol). DTPA (15.7 mg) and DL-GLA (356.7 mg) were added to 36 mL of the above solution and dissolved with a vortex mixer. Then, Ac-Lys-OH (799.6 mg) was added and stirred until it was dissolved (DTPA concentration in solution: 1 mM, DL-GLA concentration: 110 mM, Ac-Lys-OH concentration: 118 mM). Furthermore, the lid of the tube was sealed with a film such as Parafilm ™ to prevent the liquid in the tube from evaporating, and the tube was incubated at 37 ° C. for 9 days. In addition, 260 mg and 196 mg of DL-GLA were added to the reaction solutions on the 3rd and 6th days of the reaction and dissolved. The obtained reaction solution was stored at 4 ° C.

(2) Separation and purification of Lys-hydroxy-triocidin The Lys-hydroxy-triocidin reaction solution is filtered through a syringe filter (Millex-LG, Merck) having a pore size of 0.2 μm, and the filtrate is filtered by a liquid chromatograph mass spectrometer (LC / It was used as a sample of MS). The LC unit and the MS unit of the LC / MS were configured in the same manner as in Example 12. The mobile phase was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. in HPLC grade. The mobile phase A was a 10 mM ammonium acetate aqueous solution obtained by mixing 10 mL of a 1 M ammonium acetate solution and 990 mL of distilled water, and the mobile phase B was acetonitrile. At an analysis time of 0-8 minutes, the cells flowed down with a concentration gradient of mobile phase A: B = 97: 3-95: 5. It flowed down with a concentration gradient of mobile phase A: B = 95: 5-10: 90 at an analysis time of 8-10 minutes and mobile phase A: B = 10:90 at 10-13 minutes. The column used was ZORBAX SB-C18 (150 x 9.4 mm, Agilent Technologies) and the flow rate was set to 4 mL / min. 50 μL of the filtered sample solution was injected into the LC / MS apparatus, and the peak of Lys-hydroxy-triocidin was detected by a diode array detector (269 nm) and MS (cation detection). At an analysis time of 5.7 minutes, a UV-absorbing peak was observed, which contained cations at m / z = 467.3. Therefore, the peak with a retention time of 5.7 minutes was recovered by performing MS preparative with m / z = 467 as a trigger. The solution after separation was concentrated to dryness with a rotary evaporator (Tokyo Rika Kikai). The resulting precipitate was stored at −20 ° C.

(3) Confirmation of structure of Lys-hydroxy-triocidin The obtained precipitate (10 mg) was dissolved in heavy water (0.6 mL, Wako Pure Chemical Industries, Ltd.), and a sample tube for nuclear magnetic resonance (NMR) having an outer diameter of 5 mm ( Moved to Shigemi). After that, the structure of Lys-hydroxy-triocidin was confirmed by an NMR apparatus (AVANCE III HD, 500 MHz, CryoProbe mounted type, Bruker). The attribution of total hydrogen and carbon was determined from ¹ H-NMR and ^{13 C-NMR spectrum data. Further, 1 H- 1 H COSY, 1} H- 13 C HSQC, 1 H- 13 C HMBC spectrum by analyzing the confirmed the validity of the assignment. The structure of Lys-hydroxy-triocidin is shown below.

[Example 18] Reactivity evaluation of novel monoclonal antibody and GAL691 and Lys-hydroxy-triocidin by competitive ELISA (1) Reagent preparation The following reagent was dissolved in RO water to make 1 L, and used as a coating solution.
Sodium carbonate (Fujifilm Wako Pure Chemical Industries) 1.59g
Sodium hydrogen carbonate (Fujifilm Wako Pure Chemical Industries, Ltd.) 2.93 g
BSA (5 g, Sigma-Aldrich) was dissolved in PBS (500 mL) and used as a blocking solution.

50 mM Tris (6.1 g, Wako Pure Chemical Industries, Ltd.) was dissolved in RO water (about 900 mL), and the pH was adjusted to 7.4 with 6N hydrochloric acid (Wako Pure Chemical Industries, Ltd.). Glycerol (1 mL, Sigma-Aldrich) and Tween20 (1 mL, Nacalai Tesque) were added to the obtained solution, and the volume was increased to 1 L with RO water. The resulting solution was used as a diluent.

The following reagents were dissolved in RO water to make 1 L, and 9 L of RO water was further added, and then Tween 20 (5 mL) was added. The obtained solution was used as a washing solution.
Sodium chloride (Fujifilm Wako Pure Chemical Industries) 80g
Potassium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) 2g
Disodium hydrogen phosphate dodecahydrate (Fujifilm Wako Pure Chemical Industries, Ltd.) 29 g

(2) Immobilization of antigen A solution of glyceraldehyde-derived AGEs-containing BSA (stock solution: 10 mg / mL PBS solution) prepared at 1 μg / mL in the coating solution was added to a 96-well microtiter plate (COSTAR) of 100 μL each. Incubated overnight at 4 ° C.

(3) Blocking Each well treated for solid phase was washed 3 times with a washing solution (300 μL), a blocking solution (200 μL) was added, and the mixture was left at room temperature for 1 hour.

(4) Competitive Experiment GAL691 (prepared in Example 16) and Lys-hydroxy-triocidin (prepared in Example 17) were dissolved in phosphate buffer (0.2M, pH 7.4), respectively, and 10 mg / mL. The solution was prepared. Then, by diluting with a diluted solution, a sample solution of a 2-fold dilution series (5 points) of 0.625 to 10 mg / mL was prepared. A diluted solution was prepared in the same manner for the blank solution containing no sample. A POD-labeled novel monoclonal antibody (SJ-5) solution was diluted 12,000 times with a diluted solution containing BSA (1 mg / mL, Fujifilm Wako Pure Chemical Industries, Ltd.) to obtain a POD-labeled SJ-5 antibody diluted solution.

Each well treated with the blocking solution was washed 3 times with a wash solution (300 μL), a 2-fold dilution series of sample solution (50 μL each) and a POD-labeled SJ-5 antibody dilution solution (50 μL) were added, and 2 with a plate mixer. After stirring for 1 minute, the mixture was incubated at 25 ° C. for 1 hour.

(5) Color development After washing 3 times with a washing solution (300 μL), 100 μL of a substrate solution (ELISA POD substrate TMB kit (Popular), Nacalai Tesque) was added, and the mixture was incubated at room temperature for 10 minutes under shading. Then, 2N sulfuric acid (50 μL) was added to stop the color development.

(6) Absorbance measurement and data analysis The absorbance at the main wavelength of 450 nm and the sub-wavelength of 650 nm was measured with a microplate reader (Cytion5, BioTek), and the absorbance of the sub-wavelength was subtracted from the absorbance of the main wavelength. The change in absorbance of GAL691 and Lys-hydroxy-triocidin with respect to the absorbance of the blank solution was expressed as a relative value. The result is shown in FIG. In GAL691, the absorbance decreased in a concentration-dependent manner, but in Lys-hydroxy-triocidin, no decrease in absorbance was observed. Therefore, it was revealed that the novel monoclonal antibody SJ-5 binds to the glyceraldehyde-derived AGEs of the novel structure and does not recognize Lys-hydroxy-triocidin, which is a known glyceraldehyde-derived AGEs.

[Example 19] Radical measurement of GAL691 and known Glycer-AGEs GAL691 (10 mg / mL PBS solution) prepared in Example 16 was analyzed for radicals by ESR. The ESR measurement conditions are the same as in Example 10 except for the following parameters:
Sampling time: 0.06s;
Field modulation Amplitude: 0.2mT; and
Microwave power: 15mW.
The magnetic field was corrected by the g value (2.00264) of α, γ-bisdiphenylene-β-phenylallyl (BDPA).

From the ESR spectrum of FIG. 20A, a signal indicating an organic radical of g = 2.00328 was detected in GAL691. This signal was greatly attenuated by the addition of 10% (w / v) sodium sulfite (Na ₂ SO ₃ , Fujifilm Wako Pure Chemical Industries, Ltd.) (Fig. 20B). Further, GLAP (Example 12) and Lys-hydroxy-triocidin (Example 17) (PBS solution of 10 mg / mL each) were also tested in the same manner for ESR measurement, but no signal derived from a radical could be detected (Example 12). 20C, 20D). From the above results, it was confirmed that GAL691 has radical properties not found in known Glycer-AGEs.

[Example 20] Effect of light irradiation on the oxidative activity of GAL691 AGEs are accumulated in tissues such as the vitreous body, Bruch membrane, and crystalline lens of the eye, and are used for various diseases including diabetic retinopathy and age-related macular degeneration. It has been pointed out that it is involved (Yokoi et al., Br. J. Ophthalmol., 2005, 89, 673-675; Glenn et al., Invest. Ophthalmol. Vis. Sci., 2009, 50 (1), 441-451; Nagaraj et al., Amino Acids, 201242, 1205-1220; Katagiri et al., Int. Ophthalmol., 2017, 38 (2), 1-9; Kanda et al., Sci. Rep., 2017 , 7, 16168). The eye is also an important organ that transmits information to the brain by converting light into electrical signals. Therefore, paying attention to the relationship between AGEs and light, the effect of light on the oxidative activity of GAL691 was examined.

The oxidative activity of GAL691 and Z-Lys (2 mg / mL PBS solution each) prepared in Example 16 was examined in the same manner as in Example 11. At this time, the reaction was carried out overnight under irradiation with a white LED (3000 lux).

As shown in FIG. 21, the product due to the oxidation of DAB increased 2.6 times in the treatment under GAL691 and light irradiation as compared with the treatment with GAL691 alone (under shading). Therefore, it was revealed that the oxidizing activity of GAL691 becomes stronger under light irradiation.

[Example 21] Generation of singlet oxygen by photosensitizing action of GAL691 The ability of GAL691 prepared in Example 16 to generate singlet oxygen by photosensitizing action was verified. N-α, N-ε-di (carbobenzoxi) -L-lysine (Z-Lys (Z) -OH) used as a control group of GAL691 was purchased from Watanabe Chemical Industry.

In a 0.6 mL microtube (Watson), 25 μL of GAL691 or Z-Lys (Z) -OH (10 mM, PBS solution), 12.5 μL of 4-hydroxy-2,2,6,6-tetramethylpiperidine (4-OH-TEMP; Tokyo Chemical Industry, 400 mM, PBS solution), 12.5 μL of PBS was mixed to prepare a reaction solution. 4-OH-TEMP captures singlet oxygen and converts it into nitroxide radicals, so it is often used for detection of singlet oxygen using ESR (Nakamura et al., J. Clin. Biochem. Nutr., 2011, 49 (2), 87-95). Using a portable LED light source (Mitate Imaging), the reaction solution was irradiated with light of UV 365 nm, blue 470 nm, green 530 nm, or red 630 nm for 1 hour. Then, ESR was measured using a part (50 μL) of each reaction solution. The ESR measurement was carried out under the same conditions as in Example 10 except for some parameters. The changed measurement parameters are as follows:
Field center: 3354.47G; Field width: 70G; Sampling time: 0.06s; Field modulation amplitude: 0.1mT; Microwave power: 15mW.

The following analysis was performed to quantify the concentration of nitroxide radicals generated. First, the spectrum of the observed nitroxide radical simulated by SpinFit module Xepr in software (Bruker), were identified radical species _{(g = 2.0058, I = 1} , A N = 17.05G). Then, the spin concentration (μM) of the simulated nitroxide radical was determined by the SpinCount module.

As shown in FIG. 22, it was found that GAL691 remarkably produces nitroxide radicals, especially when irradiated with blue light. Therefore, it was shown that GAL691 is glyceraldehyde-derived AGEs that generate singlet oxygen by photosensitization.

[Example 22] Comparison of singlet oxygen production through photosensitization between GAL691 and known glyceraldehyde-derived AGEs GAL691 (Example 16), GLAP (Example 12), Lys-hydroxy-triocidin (Example 22) For Example 17), the amount of singlet oxygen produced under blue light irradiation was compared. The sample preparation method, light source, ESR analysis conditions, and ESR spectrum analysis conditions were in accordance with Example 21. In the preparation of the measurement sample, astaxanthin (Fujifilm Wako Pure Chemical Industries, Ltd.), which is a scavenger of singlet oxygen, prepared a 10 mM dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries, Ltd.) solution, and added 12.5 μL instead of PBS. (Final concentration 2.5 mM). For astaxanthin-untreated samples, 12.5 μL of DMSO was added instead of PBS.

The result is shown in FIG. The amount of singlet oxygen produced by blue light irradiation was 9 times higher in GAL691 than in GLAP and Lys-hydroxy-triocidin. In addition, GAL691 and singlet oxygen generated by light irradiation were captured by astaxanthin. From these facts, it was shown that GAL691 is a novel glyceraldehyde-derived AGE showing a strong photosensitizing effect as compared with known glyceraldehyde-derived AGEs.

[Example 23] Examination of superoxide anion-producing ability of GAL691 For GAL691 (Example 16), GLAP (Example 12), and Lys-hydroxy-triocidin (Example 17), generation of superoxide anion under blue light irradiation. The amounts were compared. The formation of superoxide anion was confirmed by measuring ESR with 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PPH). That is, since PPH trapped in superoxide anion changes to stable nitrogen oxide, its radical concentration was measured by ESR and evaluated as the amount of superoxide anion (Dikalov et al., Biochemical and Biophysical Research Communications, 1998, 248, 211-215). A 10 mM PBS solution (pH 7.4) was prepared for PPH-HCl (Enzo Life Sciences). Each 25 μL of GAL691, Z-Lys (Z), GLAP, Lys-hydroxy-triocidin (10 mM PBS solution) was added to a 0.6 mL microtube. Then, 12.5 μL of PPH solution and 12.5 μL of PBS containing DTPA (0.4 mM) were added to prepare a total of 50 μL of the reaction solution. Then, blue light irradiation was performed for 1 hour, and ESR was measured. The light source, ESR analysis conditions, and ESR spectrum analysis conditions were the same as in Example 21, but only the Microwave power was changed to 5 mW.

The results are shown in FIG. When not irradiated with light (under shading), GAL691 produced four times or more of the amount of superoxide anion as compared with other glyceraldehyde-derived AGEs. Furthermore, the amount produced was increased about 3 times by irradiation with blue light. Therefore, it was shown that GAL691 is a glyceraldehyde-derived AGE that produces a superoxide anion independently of light, and its production amount is enhanced by light.

[Example 24] Preparation of immune antigen Glycer-AGEs-Z-Lys obtained as a precipitate in Example 2 was dissolved in 0.2 M phosphate buffer (pH 7.4) to prepare a sample solution of 50 mg / mL. bottom. Then, the mixture was filtered through a syringe filter (Millex-LG, Merck) having a pore size of 0.2 μm, and the filtrate was used as an LC / MS sample. The LC / MS analysis conditions are the same as in Example 16. Fractions with a retention time of 10-16 minutes, including the new structure GAL691, were fractionated. Acetonitrile contained in the preparative solution was removed by a rotary evaporator (Tokyo Rika Kikai). A 10% TFA aqueous solution (1 mL) was added to the residue solution (5 mL), and the mixture was inverted and mixed. Then, it was centrifuged (12,000 × g) for 10 minutes at room temperature, and the supernatant was removed. Ultrapure water (5 mL) was added to the obtained precipitate, and centrifugation was performed again to remove the supernatant. The precipitate was washed by performing this operation a total of three times. After washing, the precipitate was air-dried, the dry weight was measured, and the precipitate was stored in a refrigerator. A 20 mg precipitate was obtained per 1 mL of 50 mg / mL sample solution.

The dried precipitate was dissolved in 0.2 M phosphate buffer (pH 7.4) to give a final concentration of 40 mg / mL (A-peak solution). Further, this solution was diluted 10-fold with phosphate buffered saline (PBS, pH 7.4) to a final concentration of 4 mg / mL. The carrier protein solution was prepared as follows. A lyophilized powder of bovine serum albumin (BSA, Sigma-Aldrich) was dissolved in PBS to a final concentration of 10 mg / mL. In 1.5 mL of Eppendorf Safe-Lock Tubes, 4 mg / mL A-peak solution (500 μL) and 10 mg / mL BSA solution (200 μL) were mixed. Subsequently, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo Scientific) was dissolved in ultrapure water to prepare a 10 mg / mL aqueous solution. A 10 mg / mL EDC aqueous solution (100 μL) was quickly added to a mixed solution of A-peak and BSA (700 μL), mixed by inversion, and then incubated overnight at room temperature.

Immediately after completion of the reaction, the reaction solution was gel-filtered with Zeba Spin Desalting Columns (7K MWCO, Thermo Scientific). Furthermore, using a Slide-A-Lyzer MINI Dialysis Device (3.5K MWCO, Thermo Scientific), dialysis was performed at a low temperature (4 ° C.). The external dialysis solution was PBS. Three hours after the start of dialysis, the external fluid was exchanged, and dialysis was further performed overnight.

After completion of dialysis, the solution in the dialysis device was collected and concentrated with Amicon Ultra-0.5 (30K MWCO, Merck). The protein concentration of BSA (A-peak-BSA) in which A-peak was crosslinked was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific), and adjusted to an arbitrary protein concentration with PBS. A-peak-BSA was used as an immune antigen in the production of mouse monoclonal antibodies.

[Example 25] Preparation of monoclonal antibody (PB-1)
(1) Immunity to mice As an immune antigen, A-peak-BSA (4 mg / mL) is mixed with the same volume of Adjuvant Complete Freund (BD) to prepare an emulsion (antigen concentration: 2 mg / mL), and BALB Initial immunization was performed subcutaneously on the back of 10 / c mice (antigen amount: 200 μg / animal). Boost immunization (antigen concentration) every other week using an emulsion (antigen concentration: 0.5 mg / mL) prepared by mixing A-peak-BSA (1 mg / mL) with the same volume of Adjuvant Complete Freund (BD). Amount: 50 μg / animal) was performed. After immunization 6 times from the initial immunization, blood was collected from the tail vein to confirm the antibody titer. The antigen for booster immunization (antigen amount: 50 μg / animal) was finally administered intraperitoneally to the mouse having a high antibody titer, and 3 days later, the spleen was removed for cell fusion.

(2) Measurement of antibody titer The titer of antiserum was evaluated by ELISA. The immune antigen A-peak-BSA was added to a 96-well microtiter plate (NUNC) at a concentration of 5 μg / mL at a concentration of 50 μL / well, and solid-phased at 4 ° C. overnight. After washing 3 times with PBS (PBS-T) containing 0.05% (v / v) Tween 20, blocking was performed with PBS-T containing 0.5% (w / v) gelatin for 1 hour. The antiserum was serially diluted 500 to 5 times, and after preparing a dilution series up to 156,500 times, 50 μL / well was placed on an antigen-immobilized plate and allowed to stand for 1 hour. After washing, 50 μL / well of secondary antibody (Horseradish peroxidase (HRP) -labeled anti-mouse IgG (KPL)) diluted 10000 times with 0.1% gelatin PBS-T was added and allowed to stand for 1 hour. An o-phenylenediamine solution (100 μL) adjusted to 0.5 mg / mL with 0.1 M phosphate buffer (pH 5.0) containing 0.02% (w / v) hydrogen peroxide in each well. Was added and allowed to stand at 25 ° C. for 20 minutes, then a 2N sulfate solution (50 μL) was added to each well to stop the color development reaction. Then, the absorbance at 490 nm was measured with a microplate reader. As a result, the anti-antibody Mice that showed significant reactivity with the antigen in a diluted solution of 12500 times or more of serum were used for cell fusion.

(3) Preparation of spleen cells and cell fusion The spleen extracted from the mouse was ground to prepare ^{about 1 × 10 8 spleen cells per mouse.} P3U1 which is a myeloma cell was cultured, and P3U1 having a viable cell rate of 95% or more was prepared on the day of cell fusion. The spleen cells and P3U1 were mixed at a ratio of 5: 1 (cell number ratio), and cell fusion was performed with polyethylene glycol having a molecular weight of 4000 at a concentration of 50% (w / v). After the fusion, the cells were washed with a medium, suspended in a HAT medium, ^{and the cells were seeded into each well of a 96-well culture plate so as to be 1 × 10 5} cells / well, and a hybridoma was selectively cultured. On the 10th day of cell fusion, the hybridoma culture supernatant was collected, and the antibody titer in the culture supernatant was measured.

(4) Screening of antibody production positive wells The culture supernatant on the 10th day after cell fusion was collected, and antibody production positive wells were screened by the above antibody titer measurement method. A-peak-BSA, glyceraldehyde-derived AGEs-containing KLH (Glycer-AGEs-KLH) -positive and BSA-negative clones were selected. The method for preparing Glycer-AGEs-KLH is shown in Example 27.

(5) Cloning A clone having high specificity for A-peak-BSA was cloned by the limiting dilution method. That is, cells were prepared at 10 cells / mL in RPMI medium containing 10% FBS, and 200 μL was added to each well of two 96-well culture plates. After 10 days, it was confirmed that A-peak-BSA, Glycer-AGEs-KLH and BSA were negative in the culture supernatant, and clones derived from each well were obtained. As an antibody of the present invention having sufficient specificity, a novel monoclonal antibody PB-1 producing cell was obtained.

(6) Purification of antibody The cells producing the novel monoclonal antibody PB-1 were cultured in RPMI medium containing 10% FBS, washed with PBS, and in serum-free medium (SFM medium, GIBCO) for 5 to 7 days. It was cultured to obtain a culture supernatant. The IgG fraction was purified from the culture supernatant on a protein G column (GE Healthcare) to obtain a novel monoclonal antibody PB-1 as an antibody of the present invention having sufficient specificity.

[Example 26] Measurement of dissociation constant of monoclonal antibody PB-1 The dissociation constant (Kd value) of the monoclonal antibody PB-1 was measured by the following method. A-peak-BSA was diluted with 10 mM sodium acetate solution (pH 5.0) to prepare a ligand solution with a final concentration of 50 μg / mL. Biacore T200 (GE Healthcare) was used for ligand immobilization and Kd value calculation. The ligand solution was immobilized on the sensor chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare) (final immobilization amount 72RU). Subsequently, a 0.1953 to 50 nM PB-1 antibody diluted solution was prepared with HBS-P + (GE Healthcare) as a running buffer, and sensorgrams were obtained.

As a result of using 1: 1 binding as a fitting model and analyzing the sensorgram, the Kd value of the PB-1 monoclonal antibody was 4.15 nM.

The amino acid sequence of the variable region determined by a conventional method is shown in FIG. The underlined part represents the CDR sequence.

[Example 27] Preparation of glyceraldehyde-derived AGEs-containing scallop hemocyanin (Glycer-AGEs-KLH) The preparation method of Glycer-AGEs-KLH was based on the preparation method of Glycer-AGEs-BSA described in Example 1. .. However, instead of 500 mg of BSA, 167 mg of hemocyanin and Sukashigai (KLH, Fujifilm Wako Pure Chemical Industries, Ltd.) were reacted with DL-glyceraldehyde (KLH concentration in solution: 8.3 mg / mL).

[Example 28] Preparation of glycolaldehyde-derived pyridinium compound (GA-pyridine) GA-pyridine can be prepared by a known method (Murakami et al. Bioscience Biotechnology and Biochemistry, 2018, 82 (2), 312-319. ). Specifically, GA-pyridine was prepared by the following method.

(1) GA- pyridine Preparation of the reaction solution N ^alpha - acetyl -L- lysine (Ac-Lys-OH, Tokyo Kasei Kogyo), glycolaldehyde dimer (GA, Sigma-Aldrich) were obtained by purchase. 0.96 g of GA (0.2 M) was added to a 50 mL centrifuge tube and completely dissolved in 40 mL of phosphate buffer (0.2 M, pH 7.4). Then, 0.75 g of Ac-Lys-OH (0.1 M) was added and stirred until dissolved. The lid of the tube was sealed with a film such as Parafilm ™ to prevent the liquid in the tube from evaporating, and the tube was incubated at 50 ° C. for 3 days. The obtained reaction solution was stored at 4 ° C.

(2) Separation and Purification of GA-Pyridine The GA-pyridine reaction solution is filtered through a syringe filter (Millex-LG, Merck) having a pore size of 0.2 μm, and the filtrate is sampled by a liquid chromatograph mass spectrometer (LC / MS). And said. The LC unit and the MS unit of the LC / MS were configured in the same manner as in Example 12. The mobile phase was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. in HPLC grade. The mobile phase A was a 0.1% formic acid aqueous solution in which 1 mL of formic acid and 1000 mL of distilled water were mixed, and the mobile phase B was a 50% acetonitrile / 0.1% formic acid solution in which 500 mL of acetonitrile, 500 mL of distilled water and 1 mL of formic acid were mixed. At an analysis time of 0-10 minutes, the cells flowed down with a concentration gradient of mobile phase A: B = 100: 0-90: 10. It flowed down with a concentration gradient of mobile phase A: B = 90: 10-0: 100 at an analysis time of 10-12 minutes and with mobile phase A: B = 0: 100 at 12-15 minutes. The column was ZORBAX SB-C18 (150 x 9.4 mm, Agilent Technologies) at 35 ° C. and the flow rate was set to 4 mL / min. 80 μL of the filtered sample solution was injected into the LC / MS apparatus, and the peak of GA-pyridine was detected by a diode array detector (290 nm) and MS (cation detection). At 6 minutes of analysis time, a UV-absorbing peak was observed, which contained cations at m / z = 297.1. Therefore, the peak with a retention time of 6 minutes was recovered by performing MS preparative with m / z = 297 as a trigger. The solution after separation was concentrated to dryness with a rotary evaporator (Tokyo Rika Kikai). The resulting precipitate was stored at −20 ° C.

(3) Confirmation of structure of GA-pyridine The obtained precipitate (4.7 mg) was dissolved in heavy water (0.6 mL, Wako Pure Chemical Industries, Ltd.), and a sample tube for nuclear magnetic resonance (NMR) having an outer diameter of 5 mm ( Moved to Shigemi). After that, the structure of GA-pyridine was confirmed with an NMR device (AVANCE III HD, 500 MHz, CryoProbe mounted type, Bruker). The obtained ¹ H-NMR spectral data was compared with non-patent literature (Nagai et al., Journal of Biological Chemistry, 2002, 277 (50), 48905-48912) to determine the attribution of total hydrogen. In addition, the validity of attribution was confirmed by analyzing the ¹ H- ^{13 C HSQC spectrum.} The structure of GA-pyridine is shown below.

[Example 29] Preparation of glyoxal-derived lysine dimer (GOLD) GOLD (Wells-Knecht et al., J. Org. Chem. 1995, 60, 6264-6247) was prepared by the following method.

(1) Preparation of the reaction solution N ^alpha - acetyl -L- lysine (Ac-Lys-OH, Tokyo Kasei Kogyo), 40% glyoxal solution (Sigma-Aldrich) were obtained by purchase. 188 mg of Ac-Lys-OH was dissolved in phosphate buffer (0.5 M, pH 7.4, 1 mL) to prepare a 1 M solution. A 1M Ac-Lys-OH solution (500 μL), a 40% glyoxal solution (73 μL), and a 0.5 M phosphate buffer (427 μL) were mixed in a microtube and stirred with a vortex mixer. Then, the tube was sealed and heated at 95 ° C. for 5 minutes. The obtained reaction solution was stored at 4 ° C.

(2) Separation and Purification of GOLD The GOLD reaction solution was filtered through a syringe filter (Millex-LG, Merck) having a pore size of 0.2 μm, and the filtrate was used as a sample of a liquid chromatograph mass spectrometer (LC / MS). The LC unit and the MS unit of the LC / MS were configured in the same manner as in Example 12. The mobile phase was purchased from Fujifilm Wako Pure Chemical Industries, Ltd. in HPLC grade. The mobile phase A was a 0.1% formic acid aqueous solution in which 1 mL of formic acid and 1000 mL of distilled water were mixed, and the mobile phase B was a 50% acetonitrile / 0.1% formic acid solution in which 500 mL of acetonitrile, 500 mL of distilled water and 1 mL of formic acid were mixed. At an analysis time of 0-10 minutes, the cells flowed down with a concentration gradient of mobile phase A: B = 95: 5-70: 30. It flowed down with a concentration gradient of mobile phase A: B = 70: 30-0: 100 at an analysis time of 10-12 minutes and with mobile phase A: B = 0: 100 at 12-15 minutes. The column was ZORBAX SB-C18 (150 x 9.4 mm, Agilent Technologies) at 35 ° C. and the flow rate was set to 4 mL / min. 20 μL of the filtered sample solution was injected into the LC / MS apparatus, and the peak of GOLD was detected by a diode array detector (215 nm) and MS (cation detection). At an analysis time of 4.2 minutes, a UV-absorbing peak was observed, which contained cations at m / z = 411.2. Therefore, the peak with a retention time of 4.2 minutes was recovered by performing MS preparative with m / z = 411 as a trigger. The solution after separation was concentrated to dryness with a rotary evaporator (Tokyo Rika Kikai). The resulting precipitate was stored at −20 ° C.

(3) Confirmation of GOLD structure The obtained precipitate (6.7 mg) was dissolved in heavy water (0.6 mL, Fujifilm Wako Pure Chemical Industries, Ltd.), and a sample tube for nuclear magnetic resonance (NMR) with an outer diameter of 5 mm (Shigemi). Moved to. After that, the structure of GOLD was confirmed with an NMR device (AVANCE III HD, 500 MHz, CryoProbe mounted type, Bruker). ^{For 1} H-NMR and ¹³ C-NMR spectral data, the attribution of total hydrogen and carbon was determined with reference to the above literature. ^{Further, 1 H- 1 H COSY, 1} H- 13 C HSQC, 1 H- 13 C HMBC spectrum by analyzing the confirmed the validity of the assignment. The structure of GOLD is shown below.

[Example 30] Confirmation of specificity of novel monoclonal antibody PB-1 by ELISA competition method (1) Antigen immobilization 1 μg of coating solution (Example 13) is applied to each well of a 96-well microtiter plate (Coster). 100 μL of A-peak-BSA or Glycer-AGEs-BSA prepared to a concentration of / mL was added, and the mixture was immobilized at 4 ° C. overnight.

(2) Blocking After washing 3 times with PBS (PBS-T) containing 0.05% (v / v) Tween20, blocking was performed with PBS-T containing 0.5% (w / v) gelatin for 1 hour.

(3) Competitive reaction Contains bovine serum albumin (BSA) diluted to 10 μg / mL with 0.1% (w / v) gelatin-containing PBS-T, glucose-derived AGEs-containing BSA (Glu-AGEs-BSA), and fructose-derived AGEs. BSA (Fru-AGEs-BSA), glycolaldehyde-derived AGEs-containing BSA (Glycol-AGEs-BSA), glyceraldehyde-derived AGEs-containing BSA (Glycer-AGEs-BSA), Nε-carboxyethyl lysine-containing BSA (CEL-AGEs-) BSA), Nε-carboxymethyllysine-containing BSA (CML-AGEs-BSA), glyoxal-derived AGEs-containing BSA (GO-AGEs-BSA), methylglyoxal-derived AGEs-containing BSA (MGO-AGEs-BSA), A-peak-BSA 50 μL of each was added to the antigen-immobilized plate, and 50 μL of PB-1 antibody diluted to 0.05 μg / mL with PBS-T containing 0.1% (w / v) gelatin was added, and the mixture was stirred with a plate mixer. , The mixture was allowed to stand at room temperature for 1 hour.

(4) Reaction with detection antibody Peroxidase-labeled anti-mouse IgG (H + L) polyclonal antibody, F (ab') diluted to 0.05 μg / mL with PBS-T containing 0.1% (w / v) gelatin after washing. ) _Two fragments (KPL) were added to the wells in an amount of 100 μL each, and the mixture was allowed to stand at room temperature for 1 hour.

(5) Color development and absorbance measurement After washing, 100 μL of each well of the ELISA POD substrate TMB kit (Nacalai Tesque) was added, and the mixture was allowed to stand in a dark place at room temperature. After 10 minutes, 50 μL of 2N sulfuric acid was added to stop the color development. The absorbance at the main wavelength of 450 nm and the sub-wavelength of 650 nm was measured with a microplate reader (Cytion 5, BioTek), and the absorbance of the sub-wavelength was subtracted from the absorbance of the main wavelength.

As a result of the specificity of the PB-1 antibody when A-peak-BSA was used as the immobilized antigen in FIG. 26A, the PB-1 antibody when Glycer-AGEs-BSA was used as the immobilized antigen was shown in FIG. 26B. The result of the specificity of is shown. The vertical axis of the graph is represented by the change in absorbance when the control BSA is 1. Even when A-peak-BSA and Glycer-AGEs-BSA are immobilized, the specificity of the novel monoclonal antibody PB-1 is positive for Glycol-AGEs-BSA, Glycer-AGEs-BSA, and A-peak-BSA. , Glu-AGEs-BSA, Fru-AGEs-BSA, CEL-AGEs-BSA, CML-AGEs-BSA, GO-AGEs-BSA, MGO-AGEs-BSA.

[Example 31] Reactivity evaluation of novel monoclonal antibody PB-1 by competing ELISA (1) Antigen immobilization Competitive ELISA was performed using the reagent described in Example 13. A solution of A-peak-BSA prepared at 1 μg / mL in the coating solution was added to a 96-well microtiter plate (Costar) in 100 μL increments and incubated overnight at 4 ° C.

(2) Blocking Each well treated for solid phase was washed 3 times with a washing solution (300 μL), a blocking solution (200 μL) was added, and the mixture was left at room temperature for 1 hour.

(3) Competitive Experiment GAL691 (prepared in Example 16), GLAP (prepared in Example 12), Lys-hydroxy-triocidin (prepared in Example 17), GA-pyridine (prepared in Example 28) , GOLD (prepared in Example 29) and Z-Lys were dissolved in phosphate buffer (0.2 M, pH 7.4), respectively, to prepare a 100 mM solution. Then, it was diluted 10 times with a diluted solution containing BSA (1 mg / mL, Wako Pure Chemical Industries, Ltd.) to prepare a 10 mM solution. Further, a sample solution of 0.04, 0.16, 0.63, 2.5 mM 4-fold dilution series was prepared with 10% (v / v) phosphate buffer and 1 mg / mL BSA-containing diluent. .. As a blank solution containing no sample, a phosphate buffer solution was similarly diluted 10-fold with a 1 mg / mL BSA-containing diluted solution. For PB-1 antibody, a 0.1 μg / mL solution was prepared with a 1 mg / mL BSA-containing diluent.

Each well treated with the blocking solution was washed 3 times with a washing solution (300 μL), a 4-fold dilution series of sample solution (50 μL each) and a PB-1 antibody diluted solution (50 μL) were added, and the mixture was stirred with a plate mixer for 2 minutes. , Incubated for 1 hour at room temperature.

(4) Reaction with detection antibody Each well after the competitive reaction was washed 3 times with a washing solution (300 μL) and diluted to 0.05 μg / mL with a diluted solution containing 1 mg / mL BSA (H + L). ) Polyclonal antibody, F (ab') ₂ fragment (KPL) was added to each well in an amount of 100 μL, and the mixture was allowed to stand at room temperature for 1 hour.

(5) Color development After washing 3 times with a washing solution (300 μL), 100 μL of a substrate solution (ELISA POD substrate TMB kit (Popular), Nacalai Tesque) was added, and the mixture was incubated at room temperature for 10 minutes under shading. Then, 2N sulfuric acid (50 μL) was added to stop the color development.

(6) Absorbance measurement and data analysis The absorbance at the main wavelength of 450 nm and the sub-wavelength of 650 nm was measured with a microplate reader (Cytion5, BioTek), and the absorbance of the sub-wavelength was subtracted from the absorbance of the main wavelength. The changes in the absorbance of GAL691, GLAP, Lys-hydroxy-triocidin, GA-pyridine, GOLD, and Z-Lys with respect to the absorbance of the blank solution were expressed as relative values. The result is shown in FIG. In GAL691, the absorbance decreased in a concentration-dependent manner, but in other compounds, no decrease in absorbance was observed. Therefore, the novel monoclonal antibody PB-1 binds to the glyceraldehyde-derived AGEs of the novel structure, and GLAP and Lys-hydroxy-triocidin, which are known glyceraldehyde-derived AGEs, and GA-pyridine, which are known glycolaldehyde-derived AGEs, are present. It was revealed that it is an antibody that does not recognize GOLD, which is AGEs derived from or glyoxal.
Polyclonal

[Example 32] Suppression test of DAB oxidation by A-peak-BSA using PB-1 antibody (1) Preparation of reaction solution Diaminobenzidine (DAB, Dojin Kagaku) prepared a PBS solution of 5 mg / mL. The preparation of A-peak-BSA (2 mg / mL, PBS solution) is the same as in Example 24. 10 μL of DAB solution was added to a 0.6 mL microtube (Watson), and 100 μL of PBS, PB-1 antibody (1.8 mg / mL), or mouse IgG isotype control (Ctrl-mIgG, Thermo Fisher Scientific) was added. .. After stirring with a vortex mixer, 10 μL of A-peak-BSA solution was added. These solutions were stirred with a vortex mixer and then irradiated with a white LED (3000lux) overnight. The solution after light irradiation was stirred with a vortex mixer and then spun down, and 100 μL was collected on a Violamo 96-well plate (As One).

(2) Absorbance measurement and data analysis The absorbance at a wavelength of 460 nm was measured with a microplate reader (Cytion5, BioTek), and oxidized DAB was detected. The result is shown in FIG. The vertical axis of the graph is shown as a relative value to the oxidized DAB detected in the antibody-untreated group (PBS-added group). Compared with the PBS-treated group and the Ctrl-mIgG-treated group, the oxidized DAB was significantly reduced in the PB-1 antibody-treated group. From this result, it was shown that the PB-1 antibody can suppress the oxidizing action of DAB by the novel structure by binding to the glyceraldehyde-derived AGEs of the novel structure.

[Example 33] Cross-competition between SJ-5 antibody and anti-GA-pyridine monoclonal antibody GA-pyridine, which is glycolaldehyde-derived AGEs, has been reported by Nagai et al., Journal of Biological chemistry, 2002, 277 (50), 48905-48912), a monoclonal antibody against GA-pyridine (clone name 2A2) has also been established. Therefore, the cross-reactivity of the newly established SJ-5 antibody, PB-1 antibody, and anti-GA-pyridine antibody 2A2 was examined by competing ELISA. Some reagents used in the competitive ELISA were prepared in the same manner as in Example 13.

(1) Immobilization of antigen A solution of A-peak-BSA prepared at 1 μg / mL in a coating solution was added to a 96-well microtiter plate (Costar) of 100 μL each and incubated overnight at 4 ° C.

(2) Blocking Each well subjected to the immobilization treatment was washed 3 times with a washing solution (300 μL), and 0.05% Tween 20-containing PBS (PBS-T) (200 μL) in which 0.5% gelatin was dissolved was washed. Was added, and the mixture was allowed to stand at room temperature for 1 hour.

(3) Competitive Experiment PB-1 antibody, mouse IgG isotype control (Ctrl-mIgG, Thermo Fisher Scientific), and anti-GA-pyridine antibody 2A2 (Cosmo Bio) were diluted with PBS-T containing 0.1% gelatin. A 4-fold dilution series was prepared (0.049-50 μg / mL). In addition, the HRP-labeled SJ-5 antibody (Example 4) was diluted 10000-fold with PBS-T containing 0.1% gelatin.

Each well treated with the blocking solution was washed 3 times with a wash solution (300 μL), a 4-fold dilution series of sample solution (50 μL each) and an HRP-labeled SJ-5 antibody dilution solution (50 μL) were added, and a plate mixer was used for 2 minutes. After stirring, the mixture was incubated at room temperature for 1 hour.

(4) Color development After washing 3 times with a washing solution (300 μL), 100 μL of a substrate solution (ELISA POD substrate TMB kit (Popular), Nacalai Tesque) was added, and the mixture was incubated at room temperature for 10 minutes under shading. Then, 2N sulfuric acid (50 μL) was added to stop the color development.

(5) Absorbance measurement and data analysis The absorbance at the main wavelength of 450 nm and the sub-wavelength of 650 nm was measured with a microplate reader (Cytion5, BioTek), and the absorbance of the sub-wavelength was subtracted from the absorbance of the main wavelength. The result is shown in FIG. In the reaction between A-peak-BSA and SJ-5 antibody, the addition of PB-1 reduced the absorbance in a PB-1 concentration-dependent manner. On the other hand, in Ctrl-mIgG and anti-GA-pyridine antibody 2A2, no decrease in absorbance was observed and no cross-competition was observed. Therefore, it was shown that the SJ-5 antibody and the PB-1 antibody are antibodies that recognize the same epitope, but the anti-GA-pyridine antibody is an antibody that does not recognize the epitope of the SJ-5 antibody.

[Example 34] Tight junction decay suppression test of retinal pigment epithelial cells (ARPE-19 cells) Retinal pigment epithelial cells restrict the movement of substances from choroidal blood vessels by forming the blood-retinal barrier due to intercellular tight junctions. Maintains retinal function. Collapse of the blood-retinal barrier is thought to be the cause of diseases such as diabetic retinopathy, and tight junctions of retinal pigment epithelial cells are extremely important in maintaining retinal function (Willermain et al., Int. J. Mol). . Sci., 2018, 19 (4), pii: E1056.). The xCELLigence RTCA DP system is a device with electrodes installed on the bottom of the well. By passing a weak current through the bottom of the well, this device can measure the impedance of the bottom of the well without affecting the function of cells. Impedance has been shown to increase with the formation of intercellular tight junctions and decrease with the collapse of tight junctions (Wittchen et al., Invest. Ophthalmol. Vis. Sci., 2011, 52 (10), 7455- 63). In this example, the effect of retinal pigment epithelial cells (ARPE-19) on tight junctions by A-Peak-BSA was confirmed using the xCELLigence RTCA DP system, and its suppression with SJ-5 antibody and PB-1 antibody. The effect was evaluated.

(1) Cell culture To D-MEM (Fujifilm Wako Pure Chemical Industries), 10% of FBS (Fujifilm Wako Pure Chemical Industries) was added, and 1% of penicillin-streptomycin (Nakalitesk) was added to prepare DMEM medium, and ARPE- 19 cells (ATCC) were cultured in DMEM medium using a 10 cm dish (corning).

(2) Measurement plate The _{xCELLigence RTCA DP system (ACEA Biosciences) was installed in the CO 2} incubator and used in a state where it was set overnight or more after installation in order to stabilize it. Immediately before cell preparation, DMEM medium was added to E-plate (ACEA Biosciences) at 50 μL / well, and the background was measured.

(3) Cell preparation The medium of ARPE-19 cells cultured in (1) above was removed, and the cells were washed by repeating the addition and removal of 10 mL of PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd.) twice. Cells were dispersed by adding 1 mL of trypsin-EDTA (Fujifilm Wako Pure Chemical Industries, Ltd.) and incubating in a CO ₂ incubator (Titec) at 37 ° C. for 3 minutes. Then, 10 mL of DMEM medium was added to neutralize the activity of trypsin. The neutralized cell dispersion was transferred to a 50 mL tube (Falcon) and centrifuged at 1500 rpm for 5 minutes using a centrifuge (Kubota). The supernatant was then discarded and the precipitate was resuspended in 1 mL DMEM medium. 10 μL of the resuspended cell dispersion was taken, mixed with 10 μL of trypan blue (NanoEnTek), and 10 μL was added to the hemocytometer (NanoEnTek). Then, the number of viable cells was counted by the auto cell counter EVE (NanoEnTek). The cell suspension was diluted with DMEM medium to 4.0 x 105 cells / mL and seeded in E-plate (ACEA Biosciences) at 80 μL / well.

(4) Addition of antibody and A-peak-BSA
The impedance of the plate was measured every 15 minutes using the xCELLigence RTCA DP system to confirm the adhesion of cells to the plate. 20 to 24 hours after cell seeding, 9 μL of PB-1 antibody (10 mg / mL), PB-1 antibody (3 mg / mL), PB-1 antibody (1 mg / mL), PB-1 antibody (0.3 mg / mL) mL), SJ-5 antibody (10 mg / mL), or PBS and 1 μL A-peak-BSA (10 mg / mL) were added. Control added 10 μL of PBS after 20-24 hours. Impedance was measured at 15 minute intervals even after the addition.

The impedance measured 10 to 12 hours after the addition of A-peak-BSA was measured, and the result of standardizing the A-peak-BSA addition group to 0 is shown in FIG. As shown in the figure, it was confirmed that the impedance was significantly reduced by the addition of A-Peek-BSA. This decrease in impedance was significantly suppressed by the addition of PB-1 antibody. In addition, in the SJ-5 antibody-added group, suppression of the list price was observed at a final concentration of 1 mg / mL, but in the addition of PB-1 antibody, stronger suppression was observed than in the SJ-5 antibody even in the lower concentration range. From the above results, it was suggested that A-peak-BSA induces the disruption of tight junctions in retinal pigment epithelial cells, which can be suppressed by the addition of PB-1 antibody and SJ-5 antibody.

[Example 35] Neutralization test for suppressing vascular endothelial cell lumen formation by Glycer-AGEs-BSA using PB-1 antibody (1) Preparation of Matrigel matrix The preparation of Matrigel matrix was carried out in the same manner as in Example 14. ..

(2) Preparation of Glycer-AGEs-BSA and antibody reaction solution 10 μL and 10 μL of Glycer-AGEs-BSA (10 mg / mL) prepared in Example 1 (PBS, Fujifilm Wako Pure Chemical Industries, Ltd.), 90 μL of PB-1 antibody (10 mg / mL) or control antibody (10 mg / mL) was added to a 1.5 mL tube (Watson) and allowed to stand at room temperature for 10 minutes. Then, it was centrifuged at 14000 rpm for 15 minutes using a centrifuge (Thermo Fisher Scientific), and the supernatant was collected.

(3) The lumen formation inhibition test was performed in the same manner as in Example 14. The morphology of HUVEC after culturing for 8 hours is shown in FIG. As shown in the figure, HUVEC was confirmed to form lumens in the Matrigel matrix in the control BSA-added group. However, Glycer-AGEs-BSA inhibited lumen formation. In addition, this inhibition of lumen formation was neutralized in the reaction solution with PB-1. From this result, it was clarified that the PB-1 antibody can neutralize the effect of Glycer-AGEs-BSA on HUVEC.

[Example 36] Retinal tissue staining of diabetic mice and control mice with PB-1 antibody (1) Rearing of mice C57BL6 / J mice (Claire Japan) on the 13th day of pregnancy were purchased to obtain pups. For diabetic mice, streptozotocin (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in physiological saline (Otsuka Pharmaceutical Industries, Ltd.) to a concentration of 5 mg / mL, and 300 μL was subcutaneously administered to male pups 2 days after birth. Two days after birth, 300 μL of physiological saline was subcutaneously administered to male pups as control mice. After living with the mother mouse for 4 weeks, weaned, and the diabetic mouse was bred in HFD32 (Claire Japan) for 8 weeks. Control mice were bred in CE-2 (Claire Japan) for 8 weeks.

(2) Anatomy 1.875 mL of Domitor (Nippon Zenyaku Kogyo), 2 mL of Midazolam (Sand), 2.5 mL of Betorfar (Meiji Seika Pharma) and 18.625 mL of physiological saline are mixed to prepare a three-kind mixed anesthesia. bottom. The body weight of the mice was measured with a weight scale (Tanita), and triple anesthesia was intraperitoneally administered at 0.1 mL / g. The effect of anesthesia was confirmed by the disappearance of the hindlimb retracting reflex, and the abdomen and thoracotomy were performed with tweezers (Natsume Seisakusho) and scissors (Natsume Seisakusho), and the inferior vena cava was amputated. 10 mL of physiological saline was administered from the left chamber for perfusion, and the eyeball was removed after perfusion. The removed eyeball was submerged in a cryomold (Sakura Finetech Japan) filled with an OCT compound (Sakura Finetech Japan) and frozen in liquid nitrogen to prepare a fresh freezing block. After preparation, it was stored in a deep freezer (Thermo Fisher Scientific) set at -80 ° C.

(3) Slicing / Dyeing Acetone (Fujifilm Wako Pure Chemical Industries, Ltd.) was incubated for 16 hours or more in a freezer (Fukushima Kogyo) set at -30 ° C to prepare cold acetone. Triton X-100 (Nacalai Tesque) was added to PBS in an amount of 0.1% to prepare PBS-T. The fresh frozen block was sliced with a cryostat CM1950 (Leica) to a thickness of 10 μm. The sliced structure was attached to MAS coated slide glass (Matsunami Glass Industry) and dried. After drying, the tissue was fixed by incubating with cold acetone for 10 minutes. After fixation, it was washed twice with PBS-T for 10 minutes. After washing, a blocking solution was prepared by diluting Fc block (BioLegend) 50-fold with TBS-T in which 5% BSA (Sigma-Aldrich) was dissolved, and blocking was performed by incubating at room temperature for 30 minutes. After blocking, a primary antibody reaction solution prepared by diluting PB-1 antibody (1 mg / mL) or control antibody (1 mg / mL) 500-fold with TBS-T in which 1% BSA was dissolved was prepared, and a refrigerator set at 4 ° C. Incubated overnight at (Fukushima Kogyo). After the primary antibody reaction, the cells were washed twice with PBS-T for 10 minutes. A secondary antibody reaction solution prepared by diluting Alexa594-labeled Goat Anti-Mouse IgG H & L (Abcam) 100-fold with TBS-T in which 1% BSA was dissolved was prepared and reacted at room temperature for 1 hour in the dark. After the secondary antibody reaction, the cells were washed twice with PBS-T for 10 minutes. Vector Shieldories was added to the stained tissue as an embedding agent and sealed with a cover glass (Matsunami Glass Industry). Then, the fluorescence of Alexa 594 of the retina was observed with a confocal laser scanning microscope (Olympus).

(4) Results The results are shown in FIG. It was revealed that the PB-1 antibody binds more strongly to the retinal ganglion cell layer, inner nuclear layer, outer nuclear layer, and retinal pigment epithelial cell layer of diabetic mice than the control mice. In addition, when the stainability of diabetic mice was confirmed with a control antibody, similar staining results could not be obtained. Therefore, it was clarified that this stainability is due to the specific binding of the PB-1 antibody by the variable region. rice field. From these results, AGE having an epitope structure of PB-1 antibody exists in vivo, and in the retina of diabetic mice, it exists in the retinal ganglion cell layer, inner nuclear layer, outer nuclear layer, and retinal pigment epithelial cell layer. It became clear. In addition, the PB-1 antibody was found to be a useful tool for immunostaining the presence of novel structures.

[Example 37] Purification of human serum protein using PB-1 antibody column and detection by Western blotting (1) Preparation of PB-1 antibody column A solvent for PB-1 antibody was used on a PD10 column (GE Healthcare). Substituted with a coupling buffer (0.2 M sodium carbonate, 0.5 M NaCl, pH 8.3), 1 mL of a 10 mg / mL PB-1 antibody solution was prepared. NHS-activated Sepharose 4 Fast Flow (GE Healthcare) was used as the coupling carrier. 10 mL of 1 mM HCl chilled in an ice bath was poured into 1 mL of NHS-activated Sepharose 4 Fast Flow, and immediately after that, it was mixed with the previously prepared 10 mg / mL PB-1 antibody solution and mixed by inversion using a rotator (TAITEC). The PB-1 antibody was immobilized on the coupling carrier by heating at 4 ° C. overnight). Then, 3 mL each of blocking buffer (0.1M Tris-HCl, pH 8.0) and washing buffer (0.1M sodium acetate, 0.5M NaCl, pH 5.0) were poured in this order, and this operation was performed in the order of 3. Repeated times. Finally, 10 mL of PBS was flowed to replace the solvent, and then the mixture was stored at 4 ° C.

(2) Purification of human serum protein using PB-1 antibody column Human serum (pool) (Cosmo Bio Co., Ltd.) was used as a purified sample. First, 10 mL of human serum (pool) is diluted 7-fold with binding buffer (0.02 M Tris-HCl, 0.03 mM NaCl, pH 7.4) and 30 mL of SP Sepharose Fast Flow (GE Healthcare) is used. , Purified according to the product manual. The obtained eluted fraction was dialyzed against a binding buffer and then purified using 3 mL of Protein G Sepharose 4 Fast Flow (GE Healthcare) according to the product manual to remove IgG. The obtained pass-through fraction was purified using 1 mL of a PB-1 antibody column as a sample. Each fraction obtained by purification was analyzed by electrophoresis (SDS-PAGE) according to a conventional method.

The results of SDS-PAGE are shown in FIG. After passing the sample (Sample in the figure) through the PB-1 antibody column (Flow Through in the figure), 25 mL of the binding buffer was repeatedly flowed twice (Wash1, Wash2 in the figure). Then, the protein bound to the PB-1 antibody column was eluted with 10 mL of elution buffer (0.1 M glycine, pH 3.0) (Elute in the figure). The eluate was immediately neutralized with 0.4 mL of neutralizing buffer (1M Tris-HCl, pH 9.0) as a result of SDS-PAGE, which removed most contaminating proteins and increased the purity of the purified sample. Shown.

(3) Western blotting using PB-1 antibody The protein purified by the PB-1 antibody column was electrophoresed according to a conventional method (SDS-PAGE), electrotransferred to a PVDF membrane, and Western blotting was performed. The antibody reaction was carried out using PB-1 antibody as the primary antibody and Peroxidase-conjugated AffiniPure Goast Anti-Mouse IgG (H + L) (Jackson ImmunoResearch) as the secondary antibody. In addition, in order to distinguish non-specific signals due to the binding of the secondary antibody, an antibody reaction using only the secondary antibody was also performed. Next, color was developed using ECL Western Blotting Detection Reagents (GE Healthcare) to detect protein bands.

No band was detected in the PVDF membrane in which only the secondary antibody was reacted. On the other hand, the band was detected only in the PVDF membrane reacted with the PB-1 antibody (Fig. 34). From this result, it was shown that Western blotting using PB-1 antibody can detect a novel structure-modifying protein present in human serum.

[Example 38] Evaluation of drug efficacy for diabetic nephropathy by systemic administration of PB-1 antibody to diabetic mice (1) Production of diabetic mice C57BL / 6J mice on the 14th day of pregnancy were purchased from Claire Japan and gave birth after childbirth. Streptozotocin (2 mg / mL) was subcutaneously administered to pups at 100 μg / mouse. When weaned at 4 weeks of age, a high-fat diet load (HFD32, Claire Japan) was started and the animals were bred on a high-fat diet until 15 weeks of age.

(2) Administration of PB-1 antibody PB-1 and control antibody were prepared with phosphate buffer (PBS, Fujifilm Wako Pure Chemical Industries, Ltd.) so as to have a concentration of 10 mg / mL. The dose of each antibody was set to 50 mg / kg, and intraperitoneal administration was performed once a week from 7 to 15 weeks of age, for a total of 8 times.

(3) Autopsy Diabetic mice were fasted for 18 hours or more and anesthetized with a three-kind mixed anesthesia. The chest and neck were disinfected with 70% ethanol, and after thoracotomy, blood was collected by cardiac puncture. After blood collection, the inferior vena cava was cut and 10 mL was manually perfused from the left chamber using a syringe filled with physiological saline (Otsuka Pharmaceutical Co., Ltd.). After that, it was promptly dissected and the presence or absence of abnormalities was visually observed in all organs and tissues.

(4) Histopathological examination After autopsy, the kidney was fixed with Superfix for paraffin section preparation and stored at room temperature. Paraffin sections were stained with hematoxylin and eosin (HE stain) and examined microscopically. Kidney HE staining of normal mice was examined microscopically at C56BL / 6J (10 weeks old).

(5) Results The results are shown in FIG. In the kidneys of the target antibody-administered group of diabetic mice, the wall of the glomerular loop of Henle was thickened, the lumen of the blood vessel was narrowed, and the stainability of the mesangial region was increased (individual Nos. 1, 3, 4: thick arrows), and the glomerulus. Increased nuclei in the body (individual Nos. 1, 3, 4: thin arrows), increased protein content in Bowman's capsule (individuals Nos. 1, 3: dotted arrows) and degeneration of proximal tubule epithelial cells (individuals) No. 1, 2, 3: arrowhead) was observed. Of these, thickening of the glomerular loop of henle wall and increased staining of the mesangial region may correspond to tuberous sclerosis found in the glomeruli of diabetic patients. In the kidneys of the PB-1 antibody-administered group, all these pathological features were clearly alleviated.

[Example 39] Preparation of PB-1 humanized antibody A PB-1 humanized antibody was prepared according to the method described in the literature (Tsurushita et al., Methods, 2005, 36, 69-83).

Asparagine in the Asn-Gly sequence is easily deamidated, and it is known that the deamidation is one of the causes of decreased antibody activity. In order to exclude the Asn-Gly sequence contained in CDR2 of the heavy chain variable region (VH) and CDR1 of the light chain variable region (VL) of the PB-1 antibody, the full-length VH and VL in which the asparagine of the sequence was replaced with serine was removed. The oligonucleotides (ChPB-1SS VH, ChPB-1SS VL) encoding the above were synthesized by a conventional method. Oligonucleotides encoding VH and VL derived from the original PB-1 antibody (including Asn-Gly sequence) were similarly synthesized (ChPB-1NN VH, ChPB-1NN VL).

VH amino acid sequence (ChPB-1SS VH) of PB-1 antibody (SG mutant) (SEQ ID NO: 26):
EVRLQQSGPELVKPGTSVKISCKAS GYSFTGYYMH WVKQSPVKSLEWIG RIIPYSGATSYNQNFKD KASLTVDKSSRTAYMDLHSLTSEDSAVYYCAR SRYYGRAPYYFDY WGQGTTLTVSS.

VL amino acid sequence (ChPB-1SS VL) of PB-1 antibody (SG mutant) (SEQ ID NO: 32):
DVVVTQTPLSLPVSLGDQASISC RSSQSIVHSSGNTYLE WYLQKPGQSPKLLIY KVSNRFS GVPDRISGSGSGTDFTLKISRVEAGDLGVYSC FQGSHVPFT FGSGTKLEIK.

For the PB-1 antibody, a humanized amino acid sequence was designed to obtain two types of VH and VL sequences. The oligonucleotide encoding the obtained amino acid sequence was synthesized. The amino acid sequence encoded by the synthesized gene is shown below (underlined part is the CDR region):

VH1 amino acid sequence of PB-1 humanized antibody (HuPB-1 VH1) (SEQ ID NO: 27):
QVQLVQSGAEVKKPGASVKVSCKAS GYSFTGYYMH WVRQAPGQRLEWIG RIIPYSGATSYNQNFKD RATLTVDTSASTAYMELSSLRSEDTAVYYCAR SRYYGRAPYYFDY WGQGTTVTVSS.

VH2 amino acid sequence of PB-1 humanized antibody (HuPB-1 VH2) (SEQ ID NO: 28): QVQLVQSGAEVKKPGASVKVSCKAS GYSFTGYYMH WVRQAPGQRLEWIG RIIPYSGATSYNQNFKD RATLTVDKSASTAYMELSSLRSEDTAVYYCAR SRYYGRAPYYFDY

VL1 amino acid sequence of PB-1 humanized antibody (HuPB-1 VL1) (SEQ ID NO: 33): DIVMTQSPLSLPVTPGEPASISC RSSQSIVHSSGNTYLE WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYSC FQGSHVPFT

VL2 amino acid sequence of PB-1 humanized antibody (HuPB-1 VL2) (SEQ ID NO: 34): DIVMTQSPLSLPVTPGEPASISC RSSQSIVHSSGNTYLE WYLQKPGQSPQLLIY KVSNRFS GVPDRISGSGSGTDFTLKISRVEAEDVGVYSC FQGSHVPFT FGGGTKVEIK.

The obtained oligonucleotides are shown below:
<ChPB-1SS VH> (SEQ ID NO: 35)
GAGGTTCGGCTGCAACAGTCTGGACCTGAGCTGGTTAAGCCTGGGACTTCAGTGAAGATCTCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTCAAGCAAAGCCCTGTGAAGAGCCTTGAGTGGATTGGACGTATTATTCCTTACAGTGGAGCTACTAGCTACAACCAGAATTTCAAGGACAAGGCCAGCTTGACTGTAGATAAGTCTTCCAGAACAGCCTACATGGATCTCCACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCGCGATACTACGGACGAGCTCCCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

<ChPB-1SS VL> (SEQ ID NO: 36)
GATGTTGTGGTCACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGTATTGTGCATAGTAGTGGAAACACCTATCTGGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGATCAGTGGAAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTTGAGGCTGGGGATCTGGGAGTTTATTCCTGCTTTCAAGGCTCACATGTTCCATTCACCTTCGGCTCTGGGACAAAGTTGGAAATCAAA

<HuPB-1 VH1> (SEQ ID NO: 37)
CAAGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTCAGGCAAGCACCTGGCCAGAGACTTGAGTGGATTGGACGTATTATTCCTTACAGTGGAGCTACTAGCTACAACCAGAATTTCAAGGACAGAGCCACCTTGACTGTGGATACCTCTGCCAGTACAGCCTACATGGAACTCTCTAGCCTGAGATCTGAGGACACTGCAGTCTATTACTGTGCAAGATCGCGATACTACGGACGAGCTCCCTACTACTTTGACTACTGGGGCCAAGGCACCACTGTCACAGTCTCCTCA

<HuPB-1 VH2> (SEQ ID NO: 38)
CAAGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTCAGGCAAGCACCTGGCCAGAGACTTGAGTGGATTGGACGTATTATTCCTTACAGTGGAGCTACTAGCTACAACCAGAATTTCAAGGACAGAGCCACCTTGACTGTGGATAAGTCTGCCAGTACAGCCTACATGGAACTCTCTAGCCTGAGATCTGAGGACACTGCAGTCTATTACTGTGCAAGATCGCGATACTACGGACGAGCTCCCTACTACTTTGACTACTGGGGCCAAGGCACCACTGTCACAGTCTCCTCA

<HuPB-1 VL1> (SEQ ID NO: 39)
GATATCGTGATGACCCAAAGTCCACTCTCCCTGCCTGTCACTCCTGGAGAACCAGCCTCCATCTCTTGCAGATCTAGTCAGAGTATTGTGCATAGTAGTGGAAACACCTATCTGGAATGGTACCTGCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGAAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTTGAGGCTGAGGATGTGGGAGTTTATTCCTGCTTTCAAGGCTCACATGTTCCATTCACCTTCGGCGGAGGGACAAAAGTGGAAATCAAA

<HuPB-1 VL2> (SEQ ID NO: 40)
GATATCGTGATGACCCAAAGTCCACTCTCCCTGCCTGTCACTCCTGGAGAACCAGCCTCCATCTCTTGCAGATCTAGTCAGAGTATTGTGCATAGTAGTGGAAACACCTATCTGGAATGGTACCTGCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGATCAGTGGAAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTTGAGGCTGAGGATGTGGGAGTTTATTCCTGCTTTCAAGGCTCACATGTTCCATTCACCTTCGGCGGAGGGACAAAAGTGGAAATCAAA

The synthesized gene was incorporated into a vector having a human-derived constant region (IgG1 heavy chain, kappa light chain) to construct 6 types of expression vectors. The combinations of VH and VL are shown below:

(1) ChPB-1NN VH / ChPB-1NN VL,
(2) ChPB-1SS VH / ChPB-1SS VL,
(3) HuPB-1 VH1 / HuPB-1 VL1,
(4) HuPB-1 VH2 / HuPB-1 VL1,
(5) HuPB-1 VH1 / HuPB-1 VL2, and (6) HuPB-1 VH2 / HuPB-1 VL2.

PB-1 chimeric antibody and PB-1 humanized antibody were expressed by the methods described in the literature (Durocher et al., Nucleic Acids Res., 2002, 30, e9). The concentration of the antibody contained in the culture supernatant was calculated, and the antigen-binding activity was evaluated by a standard ELISA method.

The ELISA antigen (A-peak-RSA) used was prepared according to the method described in Example 24. However, rabbit serum albumin (RSA, Sigma-Aldrich) was used as the carrier protein instead of BSA.

1 μg / mL A-peak-RSA diluted with 0.2 M sodium carbonate buffer (pH: 9.4) was added to the ELISA plate at 100 μL / well. After solid phase at 4 ° C. overnight, the plates were washed with wash solution (PBS containing 0.05% Tween 20) and blocked with 300 μL / well SuperBlock Blocking Buffer (Thermo Scientific). After washing the plate, test antibody diluted with SuperBlock Blocking Buffer was added at 100 μL / well. After reaction at room temperature for 1 hour, the plates were washed and 2000-fold diluted HRP-labeled anti-human IgG polyclonal antibody (Southern Biotech) was added at 100 μL / well. After allowing to stand at room temperature for 30 minutes, the plate was washed. Then, 1-Step Ultra TMB-ELISA (Thermo Scientific) was added at 100 μL / well to develop color. The color reaction was stopped with 100 μL / well of 2N sulfuric acid, and the absorbance at 450 nm was measured.

Table 3 shows the evaluation results of the binding activity to A-peak-RSA. Chimeric antibody (ChPB-1NN VH / ChPB-1NN VL) retaining the variable region of the original PB-1 mouse antibody and 4 types of PB-1 humanized at both test antibody concentrations of 50 ng / mL and 100 ng / mL. The binding activity of the antibody was shown to be comparable.

The concentration-dependent antigen binding of the PB-1 humanized antibody was evaluated by the ELISA method (Fig. 36). Similar to the results in Table 3, it was shown that the binding activity of the PB-1 chimeric antibody (ChPB-1NN VH / ChPB-1NN VL) and the four types of PB-1 humanized antibodies were equivalent.

Claims (18)

(1) The amino acid sequences of the complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3) of the heavy chain variable region or the complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3) of the light chain variable region are
(1-3H)
(A) VH CDR1: The amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence substantially the same as that;
(B) VH CDR2: Amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence substantially identical thereto; and (c) VH CDR3: Amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence substantially identical thereto. ; Or (1-3L)
(D) VL CDR1: The amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence substantially the same as that;
(E) VL CDR2: Amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence substantially the same as that; and (f) VL CDR3: Amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence substantially the same thereof. ;
A monoclonal antibody or antigen-binding fragment thereof, which comprises. (1) The amino acid sequences of the complementarity determining regions (VH CDR1, VH CDR2, and VH CDR3) of the heavy chain variable region and the complementarity determining regions (VL CDR1, VL CDR2, and VL CDR3) of the light chain variable region are
(A) VH CDR1: The amino acid sequence shown in SEQ ID NO: 23, or an amino acid sequence substantially the same as that;
(B) VH CDR2: Amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence substantially the same as that;
(C) VH CDR3: Amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence substantially the same as that;
(D) VL CDR1: The amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence substantially the same as that;
(E) VL CDR2: Amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence substantially the same as that; and (f) VL CDR3: Amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence substantially the same thereof. ;
The monoclonal antibody according to claim 1 or an antigen-binding fragment thereof. (1) The amino acid sequences of the heavy chain variable region and the light chain variable region are
The amino acid sequence of SEQ ID NO: 26 or substantially the same amino acid sequence, and the amino acid sequence of SEQ ID NO: 32 or substantially the same amino acid sequence;
The amino acid sequence of SEQ ID NO: 27 or substantially the same amino acid sequence and / or the amino acid sequence of SEQ ID NO: 33 or substantially the same amino acid sequence;
The amino acid sequence of SEQ ID NO: 28 or substantially the same amino acid sequence and / or the amino acid sequence of SEQ ID NO: 34 or substantially the same amino acid sequence;
The monoclonal antibody according to claim 1 or 2, or an antigen-binding fragment thereof. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which binds to an epitope contained in glyceraldehyde-derived AGEs. Formula (I) or (II):

[In the formula, R ¹ , R ² , R ³ and R ⁴ are independently selected from a hydrogen atom, a protecting group, and a peptide group having 1 to 1000 amino acid residues.
X ¹ and X ² represent -O-, or -NH-;
Y ¹ and Y ² are hydrogen atoms, protecting groups, or groups:

Represents;
R ⁵ and R ⁶ are independently selected from hydrogen atoms, protecting groups, and peptide groups with 1 to 1000 amino acid residues]
The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which binds to the compound, the cation radical thereof, or the dication thereof. The monoclonal antibody according to any one of claims 1 to 6, which is a full-length antibody, Fab, Fab', F (ab') ₂ , Fv, scFv, dsFv, diabody, or sc (Fv) _2. The antigen-binding fragment. The monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, which is a mouse antibody, a humanized antibody, a human antibody, a chimeric antibody, or an antigen-binding fragment thereof. The monoclonal antibody or antigen thereof according to any one of claims 1 to 7, which binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs with a dissociation constant (K _d value) of 1 × ^{10-5 M or less.} Bonded fragment. A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 1 to 8 or an antigen-binding fragment thereof. Obesity, diabetes, diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic myocardium, diabetic vascular complications, diabetic nephropathy, diabetic kidney disease, diabetic foot lesions, diabetic ketoacidosis, periodontal disease, age-related yellow spot degeneration Disease, pulmonary fibrosis, idiopathic pulmonary fibrosis, peribronchial fibrosis, interstitial lung disease, lung cancer, cancer fibrotic lung disease, chronic obstructive pulmonary disease, acute lower extremity arterial embolism, peripheral arterial disease, peripheral Airway disease, pulmonary emphysema, nephritis, glomerulosclerosis, glomerulonephritis, mesangial proliferative glomerulonephritis, diabetic nephropathy, renal systemic fibrosis, chronic kidney disease, idiopathic retroperitoneal fibrosis, renal disease, strong skin Disease, renal stromal fibrosis, female infertility, polycystic ovary syndrome, ovarian dysfunction, early ovarian dysfunction, ovarian cancer, breast cancer, uterine body cancer, prostate cancer, male infertility, liver disease, liver cirrhosis , Non-alcoholic steatosis, liver cancer, atherosclerotic cerebral infarction, atherosclerosis, internal carotid artery stenosis, aortic valve stenosis, aortic valve insufficiency, cardiovascular disease, angina, congestive Heart failure, acute heart failure, chronic heart failure, ischemic heart disease, dilated cardiomyopathy, cardiac sarcoidosis, hypertension, pulmonary arterial pulmonary hypertension, pulmonary heart, myocarditis, vascular stenosis heart fibrosis, post-myocardial infarction heart fibrosis, myocardium Post-infarction left ventricular hypertrophy, rheumatoid arthritis, lifestyle disease, dyslipidemia, Alzheimer's disease, vascular dementia, cerebral infarction, brain tumor, cerebrovascular disorder, melanitis, endocrine disease, osteoporosis, tongue cancer, oral cavity Cancer, pharyngeal cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, retinal pigment degeneration, diabetic luteal edema, Labor congenital melanosis, Stargart's disease, Asher syndrome, colloideremia, rod cone Body dystrophy, pyramidal dystrophy, progressive retinal atrophy, luteal dystrophy, choroidal sclerosis, total choroidal atrophy, cystic edema, vasculitis, retinal detachment, luteal foramen, luteal capillary dilatation, glaucoma, The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is used for diagnosing, treating, or preventing a disease selected from optic neuropathy, ischemic retinal disease, premature infant retinosis, retinal vascular occlusion, and retinal aneurysm. Diabetes mellitus, impaired glucose tolerance, retinopathy, nephropathy, complications associated with diabetes, peripheral neuropathy, lower limb necrosis, arteriosclerosis, thrombosis, non-alcoholic fatty liver disease, non-alcoholic steatosis, cancer, infertility The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is used for diagnosing, treating, or preventing a disease selected from neurodegenerative diseases including polycystic ovary syndrome, ovarian dysfunction, central neuropathy, and Alzheimer's disease. The pharmaceutical composition according to claim 11, wherein the disease is cancer selected from melanoma, lung cancer, and liver cancer. Claims for use in the diagnosis, treatment, or prevention of diseases selected from diabetic nephropathy, pyelonephritis, glomerulonephritis, glomerulonephritis, renal systemic fibrosis, chronic kidney disease, and renal interstitial fibrosis. Item 9. The pharmaceutical composition according to Item 9. The pharmaceutical composition according to claim 9, which is used for diagnosing, treating, or preventing eye diseases. Eye diseases include diabetic retinopathy, diabetic cataract, retinal pigment degeneration, diabetic macular edema, Labor congenital melanosis, Stargart's disease, Asher syndrome, colloideremia, rod pyramidal dystrophy, pyramidal dystrophy, progressive retinal atrophy, aging Macular degeneration, macular dystrophy, choriosclerosis, total choroidal atrophy, cystic macular edema, macular inflammation, retinal detachment, macular foramen, macular capillary dilatation, glaucoma, optic neuropathy, ischemic retinal disease, The pharmaceutical composition according to claim 14, which is selected from premature infant retina, retinal vascular occlusion, and retinal macula. A nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 8 or an antigen-binding fragment thereof. An expression vector containing the nucleic acid according to claim 16. A host cell comprising the expression vector of claim 17.

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