JP7149509B2 - Antibodies against advanced glycation end products and uses thereof - Google Patents

Description

The present invention relates to monoclonal antibodies or antigen-binding fragments thereof that bind epitopes of advanced glycation end products (AGEs), particularly 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 group of amino acids, mainly lysine, is non-enzymatically modified by reducing sugars. be. AGEs produced by aging or hyperglycemia are considered to be associated with lifestyle-related diseases such as diabetic complications and arteriosclerosis. In particular, AGEs (Glycer-AGEs; Glycer-AGEs) derived from glyceraldehyde, a sugar metabolism intermediate, are also called toxic AGEs (TAGEs), and are attracting attention as biomarkers useful in diagnosis and prevention ( Non-Patent Documents 1-3).

Several structures contained in AGEs have been identified, one of which is carboxymethyllysine (Nε-(carboxymethyl)lysine, CML), in which the sugar moiety of the Amadori compound produced by the reaction of glucose and lysine is a transition metal. It has been identified as a substance that is oxidatively cleaved and formed with erythronic acid in the presence of . From the history of AGEs research, quantification of AGEs has been replaced by quantification of CML whose structure has been clarified.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 formation in vitro is believed to be oxidative cleavage of Schiff bases or Amadori compounds. CML is also known to be produced by the intramolecular Cannizzaro reaction with glyoxal (GO) and glycolaldehyde as precursors and by the autoxidation of glucose. Since CML in proteins is stable against acid hydrolysis, it can be quantified by high performance liquid chromatography (HPLC) or gas chromatography/mass spectrometry (GC/MS) (Non-Patent Document 1). .

AGEs prepared using bovine serum albumin (BSA) (AGEs-containing bovine serum albumin, AGEs-BSA) as antigens and competitive enzymes using anti-AGEs-BSA antibodies as quantification of AGEs by the enzyme immunoassay (EIA) method Starting with the -linked immunosorbent assay (ELISA) method, it was later found that these antibodies were anti-CML antibodies whose epitope was the CML structure (Non-Patent Document 1). On the other hand, antiserum was prepared using AGEs prepared by adding glyceraldehyde, glycolaldehyde, methylglyoxal, or glyoxal to rabbit serum albumin (RSA) as antigens, and the obtained antiserum was purified to produce CML non-CML. It has been reported that a binding fraction was obtained (Non-Patent Document 4). An ELISA assay has been performed using the polyclonal antibody obtained as the fraction, and 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 infertility treatment and glyceraldehyde-derived AGEs (Glycer-AGEs), and it has been reported that high levels of glycer-AGEs in the blood result in poor continued pregnancy rates. (Non-Patent Documents 1-3, 5 and 6). In such studies, ELISA assays using anti-glyceraldehyde-derived AGEs antibodies among the polyclonal antibodies described above are used (Non-Patent Documents 5 and 6).

In addition, preparation of a monoclonal antibody using glyceraldehyde-derived AGEs as an antigen has been reported (Non-Patent Document 7).

Journal of Kanazawa Medical University, 37(4): 141-161, 2012; Journal of Kanazawa Medical University, 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 Mibyou System Society, 21(1):93-96, 2015; Immunology Letters 167(2), 141-146, 2015.

An object of the present invention is to provide a monoclonal antibody having high selectivity and affinity for AGEs, particularly glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs, and an analytical method using the same. A further object of the present invention is to provide methods for diagnosing, treating and preventing diseases, particularly diagnosing, treating and preventing infertility using the monoclonal antibody.

As a result of research, the present inventors have found that a specific monoclonal antibody has good selectivity and high antigen-binding ability, and can be used for analytical, diagnostic, therapeutic and prophylactic methods. , have completed the present invention.

The present invention provides the following inventions.
[1] 1) Amino acids of each complementarity determining region (VH CDR1, VH CDR2, and VH CDR3) of the heavy chain variable region and each complementarity determining region (VL CDR1, VL CDR2, and VL CDR3) of the light chain variable region the array is
(A)
(a) VH CDR1: the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence substantially identical thereto;
(b) VH CDR2: the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence substantially identical thereto;
(c) VH CDR3: the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence substantially identical thereto;
(d) VL CDR1: the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence substantially identical thereto;
(e) VL CDR2: the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence substantially identical thereto;
(f) VL CDR3: the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence substantially identical thereto; or (B)
(a) VH CDR1: the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence substantially identical thereto;
(b) VH CDR2: the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence substantially identical thereto;
(c) VH CDR3: the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence substantially identical thereto;
(d) VL CDR1: the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence substantially identical thereto;
(e) VL CDR2: an amino acid sequence of 12 contiguous residues contained in the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence substantially identical thereto;
(f) VL CDR3: an amino acid sequence of consecutive nine residues contained in the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence substantially identical thereto;
or 2) an antibody or antigen-binding fragment thereof that cross-competes with said antibody or antigen-binding fragment for binding to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs,
A monoclonal antibody or antigen-binding fragment thereof that binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs.
[2] 1) the amino acid sequences of the heavy chain variable region and the light chain variable region are each:
the amino acid sequence of SEQ ID NO: 4 or substantially identical thereto, and the amino acid sequence of SEQ ID NO: 8 or substantially identical thereto; or the amino acid sequence of SEQ ID NO: 12 or substantially identical thereto an antibody or an antigen-binding fragment thereof, which is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence substantially identical thereto; or 2) for binding to epitopes of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs , an antibody or antigen-binding fragment thereof that cross-competes with said antibody or antigen-binding fragment thereof;
The monoclonal antibody or antigen-binding fragment thereof according to [1].
[3] The monoclonal antibody or antigen-binding fragment thereof of [1] or [2], wherein the epitope is that of glyceraldehyde-derived AGEs.
[4] any one of [1] to [3], which is a full-length antibody, Fab, Fab', F(ab') ₂ , Fv, scFv, dsFv, diabodies, or sc(Fv) ₂ A monoclonal antibody or antigen-binding fragment thereof.
[5] The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [4], which is a mouse antibody, humanized antibody, human antibody, chimeric antibody, or antigen-binding fragment thereof.
[6] The monoclonal antibody of any one of [1] to [5], which binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs with a dissociation constant (Kd value) of 1×10 ⁻⁵ M or less, or an antigen-binding fragment thereof.
[7] A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [6].
[8] Diabetes, impaired glucose tolerance, retinopathy, nephropathy, peripheral neuropathy, gangrene of the lower extremities, arteriosclerosis, thrombosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cancer, polycystic ovary syndrome , ovarian dysfunction, central nervous system disorder, and Alzheimer's disease, for use in diagnosing, treating, or preventing a disease.
[9] The pharmaceutical composition of [8], wherein the disease is cancer selected from melanoma, lung cancer, and liver cancer.
[10] A nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of any one of [1] to [6].
[11] An expression vector containing the nucleic acid of [10].
[12] A host cell containing the expression vector of [11].

FIG. 1 shows the test results of the competitive ELISA method for confirming the binding properties of the antibody (6B3) of the present invention to various AGEs. Numbers refer to the absorbance of each sample relative to the absorbance of control BSA. FIG. 2 shows the test results of the competitive ELISA method for confirming the binding properties of the antibody (11G4) of the present invention to various AGEs. Numbers refer to the absorbance of each sample relative to the absorbance of control BSA. FIG. 3 is a graph showing test results for confirming cross-competition between 11G4 and 6B3.

Among AGEs, glyceraldehyde-derived AGEs (Glycer-AGEs), also called TAGEs (Toxic AGEs), are involved in diabetes, particularly diabetic vascular complications, diabetic retinopathy, diabetic nephropathy, and diabetic macroangiopathy. has been reported. Furthermore, Glycer-AGEs are associated with hypertension, dementia such as Alzheimer's disease, cancer (e.g. liver cancer, pancreatic cancer, uterine cancer, colon cancer, rectal cancer, breast cancer, bladder cancer, malignant melanoma, and lung cancer), nonalcoholic steatohepatitis (NASH), and infertility. Glycer-AGEs can therefore be used as biomarkers for the diagnosis of these diseases, and the antibodies of the invention can be used in the detection of biomarkers.

Furthermore, the antibody according to the present invention can be used to neutralize the effects of Glycer-AGEs in vivo, and can also be used in the treatment of diseases.

In one aspect of the invention, the antibodies of the invention are isolated antibodies. Isolated antibodies do not include naturally occurring antibodies that have not been subjected to external manipulation (artificial manipulation), i.e., antibodies that are produced and reside within an individual's body. Antibodies of the invention are typically monoclonal antibodies, or antigen-binding fragments thereof.

The term "substantially identical" as used in reference to amino acid sequences means that the two amino acid sequences being compared have relatively little sequence difference and that the sequence difference has no substantial effect on specific binding to an antigen. means not giving. A substantially identical amino acid sequence may contain a partial modification of the amino acid sequence, for example, deletion, substitution, or substitution of 1 to several (eg, 1 to 3) amino acids The amino acid sequence may be modified by addition or insertion of 1 to several (eg, 1 to 3) amino acids, or a combination thereof. The position of the mutation in the amino acid sequence is not particularly limited, and multiple positions may be mutated. The number of amino acids modified in the amino acid sequence is, for example, within 10% of all amino acids shown, preferably within 5% of all amino acids. More preferably, the number is within 1% of all amino acids.

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

Naturally occurring amino acids are also classified into the following groups based on common side chain properties:
(1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile,
(2) Polar, uncharged: Cys, Ser, Thr, Asn, Gln,
(3) acidic (negatively charged): Asp, Glu,
(4) basic (positively charged): Lys, Arg, His,
(5) residues that influence chain orientation: Gly, Pro; and (6) aromatics: Trp, Tyr, Phe.

In one embodiment, substitutions may be made using amino acids belonging to the same group as the amino acids to be substituted.

Whether or not two amino acid sequences are substantially identical can be determined by comparing the antigen-binding specificities of antibodies containing each amino acid sequence (sequences in other regions are identical). For example, if the dissociation constant (Kd value) of the reference antibody for the antigen in a physiological saline environment is A, the Kd value of the antibody to be compared is in the range of A×10 ⁻¹ to A×10. Substantial identity can be recognized.

Nucleic acids according to the present invention are typically isolated nucleic acids, and in the case of nucleic acids produced by recombinant techniques, e.g. A nucleic acid that is substantially free of liquid. Similarly, the "isolated nucleic acid" in the case of a nucleic acid produced by chemical synthesis is preferably in a state substantially free of precursors (raw materials) such as dNTPs and chemical substances used in the synthesis process. 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 acids of the present invention is not limited, ie, they may be either single-stranded or double-stranded. Double-stranded DNA is preferred. Codon degeneracy is also taken into account. That is, a nucleic acid encoding a protein may have any base sequence as long as the protein can be obtained as an expression product.

AGEs are formed by the saccharification reaction of proteins. Reducing sugars such as glucose and fructose react non-enzymatically with free amino groups of proteins to form Amadori compounds from Schiff bases. This leads to the production of yellow-brown complex substances AGEs with fluorescence. On the other hand, compounds obtained by reacting amino acid residues such as lysine with sugars have been identified as structures of AGEs, and non-fluorescent pyraline, Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine ( CEL), and Nω-carboxymethylarginine (CMA) are also known as AGEs. In addition, compounds having aromatic rings such as argpyrimidine, pentosidine, crossrin, GA-pyridine, vesperlysine, and pyropyridine are specified as AGEs. AGEs also include peptides and proteins containing these compounds as amino acid residues.

Substrates for protein saccharification reactions that form AGEs include not only reducing sugars such as glucose and fructose, but also glyceraldehyde, glycolaldehyde, methylglyoxal, glyoxal, and 3-deoxyglucose, which are produced by in vivo sugar metabolism. Son and others 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), methylglyoxal-derived AGEs thus prepared Among various AGEs such as (MGO-AGEs), glyoxal-derived AGEs (GO-AGEs), and 3-deoxyglucosone-derived AGEs (3-DG-AGEs), glyceraldehyde-derived AGEs are associated with various diseases such as diabetes. It has been reported that it is involved, and a method of measuring the blood concentration of glycer-AGEs by an analysis method using a polyclonal antibody whose antigen is glyceraldehyde-derived AGEs is known.

The monoclonal antibody of the present invention has the property of specifically binding to glyceraldehyde-derived AGEs (Glycer-AGEs) and glycolaldehyde-derived AGEs (Glycol-AGEs).

In one aspect of the present invention, binding to epitopes of glyceraldehyde-derived AGEs (Glycer-AGEs) and/or glycolaldehyde-derived AGEs (Glycol-AGEs), glucose-derived AGEs (Glu-AGEs), fructose-derived AGEs (Fru -AGEs), methylglyoxal-derived AGEs (MGO-AGEs), glyoxal-derived AGEs (GO-AGEs), and 3-deoxyglucosone-derived AGEs (3-DG-AGEs). Monoclonal antibodies or antigen-binding fragments thereof are provided. More specifically, it binds to epitopes of glyceraldehyde-derived AGEs (Glycer-AGEs) and glycolaldehyde-derived AGEs (Glycol-AGEs), glucose-derived AGEs (Glu-AGEs), fructose-derived AGEs (Fru-AGEs), Monoclonal antibodies or antigen-binding fragments thereof that do not bind to methylglyoxal-derived AGEs (MGO-AGEs), glyoxal-derived AGEs (GO-AGEs), and 3-deoxyglucosone-derived AGEs (3-DG-AGEs) are provided. . More specifically, it binds to epitopes of glyceraldehyde-derived AGEs and glycolaldehyde-derived AGEs, glucose-derived AGEs (Glu-AGEs), fructose-derived AGEs (Fru-AGEs), methylglyoxal-derived AGEs (MGO-AGEs), A monoclonal antibody that does not bind glyoxal-derived AGEs (GO-AGEs), 3-deoxyglucosone-derived AGEs (3-DG-AGEs), Nε-carboxymethyllysine (CML), and Nε-carboxyethyllysine (CEL), or Antigen-binding fragments thereof are provided.

In one aspect of the invention, monoclonal antibodies or antigen-binding fragments thereof that bind to epitopes of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs are provided. Here, glyceraldehyde-derived AGEs and glycolaldehyde-derived AGEs are AGEs produced by protein glycation reactions such as BSA and mouse serum albumin (MSA) in the presence of glyceraldehyde or glycolaldehyde, and proteins containing AGEs. . The monoclonal antibodies and antigen-binding fragments thereof of the present invention are characterized by having epitopes in chemical structures produced by protein saccharification. In preferred embodiments, the antibodies and antigen-binding fragments thereof of the present invention are derived from aldehydes generated in reducing sugars and sugar metabolism, particularly glucose-derived AGEs, fructose-derived AGEs, methylglyoxal-derived AGEs, glyoxal-derived AGEs, and 3-deoxyglucosone-derived AGEs. Does not bind to AGEs. These AGEs are also prepared by protein saccharification reaction by adding each reducing sugar or aldehyde to protein such as BSA. The binding specificity of antibodies can be determined by known techniques such as competitive ELISA.

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 thereof are used for the treatment, prevention and/or diagnosis of diseases associated with AGEs. can be used. Antibodies that can be used in the present invention can take multiple forms, as described herein, and the present invention includes a set of six CDRs (amino acids substantially identical thereto) as defined herein. sequences, including sequences containing deletions, substitutions, additions of 1-3 amino acid residues, for example.

Conventional antibody structural units typically include tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair consisting of one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain. chain (typically with a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA and IgE, respectively. IgG has multiple subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. Thus, as used herein, "isotype" refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE. Therapeutic antibodies may also include isotype and/or subclass hybrids.

Each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. Three loops assemble in the variable region for each V domain of the heavy and light chains to form the antigen-binding site. Each loop is also referred to as a complementarity determining region (hereinafter also referred to as "CDR"), and amino acid sequence variation is most prominent in this region. "Variable" refers to the fact that certain segments of the variable regions differ extensively in antibody sequence. The variability within the variable regions is not evenly distributed. Alternatively, the V regions are called framework regions (FR) of 15-30 amino acids separated by short regions of extreme variability called "hypervariable regions", each 9-15 amino acids or more in length. Consists of a relatively constant stretch.

Each VH and VL is composed 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 region generally comprises about amino acid residues 24-34 (VL CDR1; "VL" means light chain variable region), about amino acid residues 50-56 (VL CDR2) and 89-97 in the light chain variable region. around (VL CDR3) and around 31-35 (VH CDR1; "VH" means the variable region of the heavy chain), around 50-65 (VH CDR2) and around 95-102 (VH CDR3) in the heavy chain variable region. ); Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and/or those that form hypervariable loops. residues such as 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; 53-55 (VH CDR2) and 96-101 (VH CDR3); Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.

Throughout this specification, when referring to residues of the variable domains (approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region), the EU numbers used for the Fc region are: The Kabat numbering system is commonly used with the system (eg Kabat et al., supra (1991)).

CDRs contribute to the formation of antigen binding, and more particularly the epitope binding site of an antibody. By "epitope" is meant a determinant that interacts with a specific antigen-binding site (paratope) in the variable region of an antibody molecule. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. As shown herein, two different antibodies, referred to herein as "6B3" and "11G4", according to the present invention have been identified to cross-compete for binding to glyceraldehyde-derived AGEs, and the same It is believed to bind epitopes.

Various AGEs can be prepared by mixing proteins (eg, bovine serum albumin (BSA), mouse serum albumin (MSA), rabbit serum albumin (RSA), etc.) with aldehydes or reducing sugars and allowing them to react. For example, glyceraldehyde-derived AGEs-containing BSA can be prepared by reacting glyceraldehyde with BSA, and this can be used as a standard of AGEs. For glyceraldehyde-derived AGEs, the antibodies of the present invention are prepared, for example, by selecting antibodies that have binding properties to BSA containing glyceraldehyde-derived AGEs and do not have binding properties to BSA. be able to. In one aspect of the present invention, the epitope to which the antibody of the present invention binds includes structures produced by non-enzymatic reactions from glyceraldehyde and proteins.

In one aspect of the present invention, there is provided an antibody, or an antigen-binding fragment thereof, comprising the amino acid sequences specified in (A) and (B) below in the complementarity determining regions of the heavy and light chain variable regions. Ru:
(A)
(a) VH CDR1: the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence substantially identical thereto;
(b) VH CDR2: the amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence substantially identical thereto;
(c) VH CDR3: the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence substantially identical thereto;
(d) VL CDR1: the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence substantially identical thereto;
(e) VL CDR2: the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence substantially identical thereto;
(f) VL CDR3: the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence substantially identical thereto; or (B)
(a) VH CDR1: the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence substantially identical thereto;
(b) VH CDR2: the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence substantially identical thereto;
(c) VH CDR3: the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence substantially identical thereto;
(d) VL CDR1: the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence substantially identical thereto;
(e) VL CDR2: an amino acid sequence of 12 contiguous residues contained in the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence substantially identical thereto;
(f) VL CDR3: an amino acid sequence of consecutive nine residues contained in the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence substantially identical thereto.

According to one aspect of the invention, the amino acid sequence of the heavy chain variable region is the amino acid sequence of SEQ ID NO:4 or substantially identical thereto, and the amino acid sequence of the light chain variable region is SEQ ID NO:8. a monoclonal antibody or an antigen-binding fragment thereof that is the amino acid sequence of or substantially identical to the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence substantially identical thereto; Antibodies or antigen-binding fragments thereof are provided, wherein the amino acid sequence of the light chain variable region is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence substantially identical thereto. In its specific aspects, the invention includes the monoclonal antibodies 6B3 and 11G4 described herein. These antibodies are mouse-derived antibodies, and have mouse antibody constant regions in addition to the heavy and light chain variable regions shown by the above SEQ ID NOs. Examples of amino acid sequences that are substantially identical to the heavy chain variable region or light chain variable region shown by the above SEQ ID NOs include sequences with one or two amino acids added to the N-terminus or C-terminus. For example, one or two amino acids (e.g., amino acids selected from naturally occurring amino acids) at the N-terminus or C-terminus as an amino acid sequence substantially identical to the heavy chain variable region shown in the above SEQ ID NO, more specifically includes sequences 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 used as a reference antibody with the above heavy chain variable regions and light chain variable regions or antibodies specified by their CDRs or antigen-binding fragments thereof. Antibodies or antigen-binding fragments thereof that cross-compete with a reference antibody for binding to an epitope of are provided. In one aspect 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 monoclonal antibody 6B3 or 11G4 as a reference antibody is provided.

Antibodies of the present invention can be confirmed for their cross-competitive properties by performing standard binding assays with Glycer-AGEs or Glycol-AGEs using, for example, monoclonal antibodies 6B3 or 11G4 as reference antibodies. Cross-competition can be confirmed by, for example, flow cytometry or cross-competition ELISA assay.

In one aspect of the present invention, pharmaceutical compositions for treating, preventing or diagnosing diseases are provided, comprising the antibodies or antigen-binding fragments thereof described above. Target diseases are not particularly limited as long as they are diseases associated with Glycer-AGEs or Glycol-AGEs. For example, diabetes, diabetic minimal vascular complications (diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy), and diabetes macroangiopathy, hypertension, dementia such as Alzheimer's disease, cancer (e.g. liver cancer, pancreatic cancer, uterine cancer, colon cancer, rectal cancer, breast cancer, bladder cancer, malignant melanoma, and lung cancer) ), nonalcoholic steatohepatitis (NASH), infertility, and the like. According to another aspect of the invention, the disease is diabetes, impaired glucose tolerance, retinopathy, nephropathy, peripheral neuropathy, gangrene of the lower extremities, arteriosclerosis, thrombosis, non-alcoholic fatty liver disease, non-alcoholic fatty liver disease. Hepatitis, cancer (such as melanoma, lung cancer, and liver cancer), polycystic ovarian syndrome, ovarian dysfunction, central nervous system disorders, and Alzheimer's disease.

In one embodiment of the present invention, the amount of Glycer-AGEs and Glycol-AGEs present in tissue (e.g., blood) of a subject is measured using the antibody or antigen-binding fragment thereof of the present invention. A diagnostic method is provided. The diagnosis here is as already described. In another aspect of the present invention, the step of measuring the amount of Glycer-AGEs and Glycol-AGEs present in a sample (for example, a blood sample obtained by blood collection) using the antibody or antigen-binding fragment thereof of the present invention. A method of analyzing a sample is provided, comprising:

An antigen-binding fragment of an antibody of the invention is, for example, Fab, Fab', F(ab') ₂ , Fv, scFv, dsFv, diabodies, or sc(Fv) ₂ . Antibodies of the invention are, for example, murine antibodies, humanized antibodies, human antibodies, or chimeric antibodies. 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, more specifically It binds to epitopes 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. Kd values for antibodies can be determined using methods well established in the art. A preferred method for determining the Kd value of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as the Biacore® system.

In one aspect of the invention, a method for producing a monoclonal antibody or antigen-binding fragment thereof that binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs is provided. The method comprises culturing host cells transfected with a nucleic acid encoding an antibody of the invention, and can be performed 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, for example, encode both the variable and constant regions of heavy and light chains, respectively. Polynucleotides may be in the form of RNA or DNA. A coding sequence that encodes a polypeptide may contain redundancy or degeneracy of the genetic code.

In some embodiments, one or more nucleic acids encoding an antibody of the invention are incorporated into an expression vector that 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 appropriate regulatory sequences (including, but not limited to, transcriptional and translational regulatory sequences, promoters, ribosome binding sites, enhancers, origins of replication, etc.) or other elements (such as selection genes). all can be operably linked as is well known in the art. In some cases, two nucleic acids are used, each in a different expression vector (e.g., a heavy chain in a first expression vector and a light chain in a second expression vector), or they are placed in the same expression vector. can be put in The design of one or more expression vectors, including the choice of regulatory sequences, may depend on factors such as choice of host cell, desired level of protein expression, and the like.

In general, any method (e.g., transformation, transfection, electroporation, infection, etc.) appropriate to the host cell of choice is used to manipulate one or more nucleic acids into one or more expression control elements. The nucleic acid and/or expression is introduced into a suitable host cell so that it is ligated (e.g., in a vector, in a construct produced by a process in the cell, integrated into the genome of the host cell), and in a recombinant host cell is produced. The resulting recombinant host cells are placed under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable non-human animal, medium supplemented with suitable salts, growth factors, antibiotics, nutritional supplements, etc.). ), thereby producing one or more encoded polypeptides. In some cases, the heavy chain is manufactured in one cell and the light chain is manufactured 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, including: 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 (e.g. Hep G2), and many others Cell lines include, but are not limited to. Non-mammalian cells, including but not limited to bacteria, yeast, insects and plants, can also be used to express recombinant antibodies. In some embodiments, antibodies can be produced in transgenic animals such as cows and chickens.

[Example 1] Preparation of AGEs AGEs can be prepared by a known method (Hum. Reprod. 26: 604-610 (2011), Mol. Med. 6: 114-125 (2000), etc.). Specifically, various AGEs were prepared by the following method.

(1) Glyceraldehyde-derived AGEs-containing bovine serum albumin (Glycer-AGEs-BSA)
Bovine serum albumin (BSA, Sigma-Aldrich), DL-glyceraldehyde (DL-GLA, Nacalai Tesque), diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA, Dojindo Laboratories) was obtained by purchase.
DL-GLA (180 mg) and DTPA (39 mg) were weighed and transferred to 50 ml culture tubes. Phosphate buffer (0.2 M, pH 7.4, 20 ml) was added to the tube and 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 obtained mixture was passed through a 0.2 μm filter in a clean bench to make a sterile solution. The lid of the tube was sealed with a film such as Parafilm (trademark) so that the liquid in the tube would not evaporate, and the tube was incubated at 37° C. for 1 week.

The resulting reaction solution (2.5 ml each) was applied to PD-10 columns (GE Healthcare, 8 columns), and phosphate buffered saline (PBS) (pH 7.0) was applied according to the protocol for PD-10 columns. 4, 3.5 ml each).

The eluate from 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, and two 14 ml tubes were cooled in advance to a volume of 2 L. It was placed in PBS and dialyzed by stirring. Dialysis was performed in a cold room (5° C.), and the dialysate (PBS) was exchanged every 24 hours for 3 days.

After dialysis, the liquids in the two dialysis tubes were combined and put into a 50 ml culture tube, and the protein amount was measured as glyceraldehyde-derived AGEs-containing BSA (Glycer-AGEs-BSA), and the amount was measured with PBS for culture. Adjusted to the required protein concentration.
A control BSA was prepared in the same manner as in Example 1 except that no DL-GLA was 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) A solution containing diethylenetriaminepentaacetic acid (5 mM, DTPA, Dojindo Laboratories) was prepared. The solution was made sterile 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 with PBS (phosphate buffered saline) for 3 days in a low temperature room (5 ° C) (during this time , PBS changed daily).

(3) BSA containing AGEs derived from glucose (Glu-AGEs-BSA) and BSA containing AGEs derived from fructose (Fru-AGEs-BSA)
25 mg/mL bovine serum albumin (BSA, Sigma-Aldrich), D-glucose (0.5 M, Wako Pure Chemical Industries) or D-fructose (0.5 M, Wako Pure Chemical Industries, Ltd.) and DTPA (5 mM, Dojindo Laboratories) were prepared, sterilized with a 0.2 μm filter, 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 against PBS in a low temperature room (5°C) for 3 days (PBS was changed daily during this period).

(4) BSA containing AGEs derived from glyceraldehyde (Glycer-AGEs-BSA), BSA containing AGEs derived from glycolaldehyde (Glycol-AGEs-BSA), BSA containing AGEs derived from methylglyoxal (MGO-AGEs-BSA), and AGEs derived from glyoxal Containing BSA (GO-AGEs-BSA)
0.1 M glyceraldehyde (Nacalai Tesque), glycolaldehyde (Nacalai Tesque), methylglyoxal (Sigma-Aldrich), or glyoxal (Sigma-Aldrich) in 0.2 M phosphate buffer (pH 7.4) , BSA (25 mg/mL) and DTPA (5 mM) were prepared. The solution was made sterile by 0.2 μm filter sterilization and incubated at 37° C. for 1 week. Incubate at 37 ° C. for 1 week under sterile 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 time , PBS changed daily).

(5) AGEs-containing BSA derived from 3-deoxyglucosone (3-DG-AGEs-BSA)
Prepare a solution containing BSA (25 mg/mL, Sigma-Aldrich), 3-deoxyglucosone (0.2 M, Dojindo Laboratories) and DTPA (5 mM) in 0.2 M phosphate buffer (pH 7.4) did. The solution was made sterile by 0.2 μm filter sterilization and incubated at 37° C. for 2 weeks. After that, low-molecular-weight unreacted substances were removed using a PD-10 gel filtration column, and further dialyzed against PBS in a low-temperature room (5° C.) for 3 days (PBS was changed every day during this period).

(6) Nε-carboxymethyllysine-containing BSA (CML-BSA)
BSA (50 mg/mL, Sigma-Aldrich), glyoxylic acid (45 mM, Wako Pure Chemical Industries) and sodium cyanoborohydride (150 mM, Sigma-Aldrich) were added in 0.2 M phosphate buffer (pH 7.4). A solution was prepared containing The solution was made sterile by 0.2 μm filter sterilization and incubated at 37° C. for 24 hours. After that, low-molecular-weight unreacted substances were removed using a PD-10 gel filtration column, and further dialyzed against PBS in a low-temperature room (5° C.) for 3 days (PBS was changed every day during this period).

[Example 3] Preparation of monoclonal antibodies specific to Glycer-AGEs (1) Immunization of mice As an immunizing antigen, the same volume of Glycer-AGEs-MSA (1 mg/ml) as Freund's Adjuvant, Complete (Sigma-Aldrich) to prepare an emulsion (concentration of antigen: 0.5 mg/mL), and 3 GANP (registered trademark) mice (Transgenic Inc.) were subcutaneously immunized for the first time (amount of antigen: 200 μg/mouse each). ). Glycer-AGEs-MSA (1 mg/ml) was boosted every two weeks with an emulsion (concentration of antigen: 0.5 mg/ml) prepared by mixing the same volume of Freund's Adjuvant, Incomplete (Sigma-Aldrich). (Amount of antigen: 50 μg/animal) was performed. After four immunizations from the initial immunization, blood was collected from the tail vein to confirm the antibody titer. For mice with a high antibody titer, an emulsion for boosting (antigen amount: 50 μg) was finally administered intraperitoneally to the mouse, and 3 days later, the spleen was removed for cell fusion.

(2) Measurement of antibody titer The titer of the antiserum was evaluated by ELISA. To a 96-well microtiter plate (NUNC), 50 μl/well of immunizing antigen glyceraldehyde-derived AGEs-containing MSA (Glycer-AGEs-MSA) was added at a concentration of 1 μg/ml, and immobilized at 4° C. overnight. . After washing three times with PBS containing 0.05% (v/v) Tween 20 (PBS-T), the cells were blocked with carbonate buffer (pH 9.5) containing 0.5% (w/v) gelatin for 1 hour. . The antiserum was serially diluted from 1000-fold to 3-fold, and after preparation of serial dilutions up to 729000-fold, 50 μl/well of each dilution was added to an antigen-immobilized plate and allowed to stand for 1 hour. After washing, 50 μl/well of a 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. 100 μL of o-phenylenediamine solution prepared to 0.5 mg/mL with 0.1 M citrate phosphate buffer (pH 5.0) containing 0.02% (w/v) hydrogen peroxide was added and incubated at 25° C. for 10 minutes. After allowing to stand for 1 minute, 100 μL of 1 M sulfuric acid solution was added to each well to stop the color reaction, and absorbance at 490 nm was measured using a microplate reader.
As a result, mice that showed significant reactivity with the antigen in a 9000-fold or more dilution of the antiserum were used for cell fusion.

(3) Preparation of Spleen Cells and Cell Fusion Spleens excised from mice were mashed to prepare about 1×10 ⁸ spleen cells per mouse. P3U1, which is a myeloma cell, was cultured to prepare P3U1 having a viable cell rate of 95% or more 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 medium, suspended in HAT medium, and seeded into each well of a 96-well culture plate at 1×10 ⁵ cells/well for selective culture of hybridomas. On day 10 of cell fusion, the hybridoma culture supernatant was collected, and the antibody titer in the culture supernatant was measured.

(4) Screening for Antibody Production-Positive Wells Ten days after cell fusion, the culture supernatant was collected, and antibody production-positive wells were screened by the method described in Example 4 below. Clones that were positive for glyceraldehyde-derived AGEs-containing MSA (Glycer-AGEs-MSA), negative for bovine serum albumin-derived carboxymethyllysine (CML-BSA), and mouse serum albumin (MSA) were selected, and the cells were collected in 24 wells. Transferred to a plate. The cells were cultured for 1 to 2 days and screened again by the method of Example 4. Finally, positive for MSA containing glyceraldehyde-derived AGEs (Glycer-AGEs-MSA), BSA derived from glycolaldehyde AGEs (Glycol- Glucose-derived AGEs-containing BSA (Glu-AGEs-BSA), Methylglyoxal-derived AGEs-containing BSA (MGO-AGEs-BSA), Glyoxal-derived AGEs-containing BSA (GO-AGEs-BSA), Nε- Carboxymethyllysine-containing BSA (CML-BSA) and mouse serum albumin (MSA) negative clones were selected.

(5) Cloning Clones with high specificity for glyceraldehyde-derived AGEs-containing MSA (Glycer-AGEs-MSA) were cloned by limiting dilution. That is, the cells were adjusted to 5 cells/mL in RPMI medium containing 10% FCS, and 200 μL of the cells were added to each well of two 96-well culture plates. After 10 days, the antibody titer against glyceraldehyde-derived AGEs-containing MSA (Glycer-AGEs-MSA) in the culture supernatant was measured by the method described in Example 4 below, and confirmed to be positive. I got a clone that does. 6B3 and 11G4 were obtained as antibodies of the present invention with sufficient specificity.

[Example 4] Screening of antibodies In each well of a 96-well microtiter plate (NUNC), glyceraldehyde-derived AGEs-containing MSA (Glycer-AGEs) prepared to 1 µg/mL with PBS (pH 7.4) as an immobilized antigen -MSA) was added in 50 µL portions and left at 25°C for 1 hour. At the same time, to confirm the specificity, BSA containing AGEs derived from glucose (Glu-AGEs-BSA), BSA containing AGEs derived from glycolaldehyde (Glycol-AGEs-BSA), BSA containing AGEs derived from methylglyoxal (MGO-AGEs-BSA), glyoxal Similarly, 50 μL each of BSA containing derived AGEs (GO-AGEs-BSA), BSA containing Nε-carboxymethyllysine (CML-BSA), and bovine serum albumin (BSA) were added and allowed to stand at 25° C. for 1 hour. Next, after washing three times with PBS (pH 7.4) containing 0.05% (v/v) Tween20 (PBS-T), PBS-T containing 0.5% gelatin (blocking solution) was added to each well. Each 200 μL was added and left at 25° C. for 1 hour. After washing, 50 μL of the undiluted culture supernatant was added to each well and allowed to stand at 25° C. for 1 hour. Next, after washing three times with PBS-T, 50 μL of 2500-fold diluted HRP-labeled anti-mouse IgG antibody (ZYMED) was added to each well and allowed to stand at 25° C. for 1 hour. Next, after washing three times with PBS-T, 0.5 mg/mL of 0.1 M citrate phosphate buffer (pH 5.0) containing 0.02% (w/v) hydrogen peroxide was added to each well. 100 μL of the o-phenylenediamine solution prepared in step 1 was added and allowed to stand at 25° C. for 10 minutes, and then 100 μL of 1M sulfuric acid solution was added to each well to stop the color reaction. Absorbance at 490 nm was then measured by a microplate reader.

6B3 and 11G4 were obtained as antibodies of the present invention with sufficient specificity. The absorbance when the control BSA is set to 1 is shown in FIG. 1 (6B3) and FIG. 2 (11G4).
The amino acid sequences of the heavy and light chain variable regions of 6B3 are the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 8, respectively, and the amino acid sequences of the heavy and light chain variable regions of 11G4 are the sequences of The amino acid sequences of No.: 12 and SEQ ID NO: 16 were confirmed.

[Example 5] Measurement of dissociation constant of monoclonal antibody The dissociation constant (Kd value) of the obtained monoclonal antibody was measured by the following method. Glyceraldehyde-derived AGEs-containing MSA (200 μg / mL, 98 μL) was immobilized on a sensor chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare), and various antibodies were added (6B3: 0.001 ~ 100 nM, 11G4: 0.8-800 nM, rabbit polyclonal antibody: 0.1-1000 nM), and the Kd value was calculated using Biacore T200 (GE Healthcare). As a result, 6B3 was 47.6 nM, 11G4 was 376 nM, and rabbit polyclonal antibody was 751 nM.

[Example 6] Cross-competition confirmation test of 6B3 and 11G4 (1) Preparation of biotin-labeled 6B3
Biotin Sulfo-OSu (3.2 mg, Dojindo Laboratories) was dissolved in RO water (145.4 μL) and used as a biotinylation solution. The following reagents were dissolved in RO water to make 1 L, and 9 L of RO water was further added to use as PBS.

Sodium chloride (Wako Pure Chemical) 80g
Potassium dihydrogen phosphate (Wako Pure Chemical) 2g
29 g of disodium hydrogen phosphate dodecahydrate (Wako Pure Chemical Industries).

The biotinylation solution (13.4 μL) was added to the 6B3 antibody (10 mg/mL, 500 μL), and the mixture was reacted at room temperature for 2 hours while mixing by inversion using a mini disc rotor (Biocraft). During the reaction, PBS (10 mL) was added to a prepacked column for purification (NAP-5, GE Healthcare) for equilibration. The reaction solution was added to the equilibrated NAP-5, and then PBS (1 mL) was added to recover the biotin-labeled 6B3 antibody.

(2) Reagent Preparation The following reagents were dissolved in RO water to make 1 L and used as a coating solution.
Sodium carbonate (Wako Pure Chemical) 1.59g
2.93 g of sodium hydrogen carbonate (Wako Pure Chemical Industries).
BSA (5 g, Sigma-Aldrich) was dissolved in PBS (500 mL) and used as blocking solution.

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

The following reagents were dissolved in RO water to make 1 L, and after adding 9 L of RO water, Tween-20 (5 mL) was added. The resulting solution was used as the wash solution.
Sodium chloride (Wako Pure Chemical) 80g
Potassium dihydrogen phosphate (Wako Pure Chemical) 2g
29 g of disodium hydrogen phosphate dodecahydrate (Wako Pure Chemical Industries).

(3) Immobilization of antigen 100 μL of a solution of glyceraldehyde-derived AGEs-containing BSA (stock solution: 10 mg/mL PBS solution) adjusted to 1.0 μg/mL in a coating solution was applied to a 96-well microtiter plate (COSTAR). and incubated overnight at 4°C.

(4) Blocking Each well subjected to immobilization treatment was washed three times with a washing solution (300 μL), a blocking solution (200 μL) was added, and left at room temperature for 1 hour.

(5) Competition experiment Using diluents, 3-fold serial dilutions of antibody (11G4) and control mouse IgG (Thermo Fisher) were prepared from 1 mg/mL in 8 points for each antibody. A biotin-labeled 6B3 antibody PBS solution (1 mg/mL) was diluted 10,000-fold with a diluent containing BSA (1 mg/mL, Wako Pure Chemical Industries, Ltd.) to obtain a biotin-labeled 6B3 antibody diluent.

Each blocking solution-treated well was washed three times with washing solution (300 μL), 3-fold serial dilutions of 11G4 and control mouse IgG (50 μL each) and biotin-labeled 6B3 antibody dilutions (50 μL) were added, and plated. After stirring for 2 minutes with a mixer, the mixture was incubated for 2 hours under horizontal shaking conditions at 30°C.

(6) Addition of secondary antibody After washing three times with a washing solution (300 μL), HRP-labeled streptavidin solution (Thermo Fisher, 1.25 mg/mL diluted 5,000-fold with diluent, 100 μL) was added. In addition, after stirring with a plate mixer for 30 seconds, incubation was carried out at 37° C. for 1 hour.

(7) Color development After washing three times with a washing solution (300 μL), 100 μL of substrate solution (ELISA POD substrate TMB solution (Easy), Nacalai Tesque) was added, stirred for 30 seconds with a plate mixer, incubated at room temperature for 30 minutes, and then 1N. HCl (100 μL) was added to stop color development.

(8) Absorbance Measurement and Data Analysis Absorbance at 450 nm was measured with a microplate reader (Cytation5, Biotek), and the competition rate was calculated from the obtained absorbance values. The results are shown in FIG. Addition of 11G4 decreased color development in a concentration-dependent manner, confirming that 11G4 cross-competes with 6B3.

Claims (12)

The amino acid sequences of each complementarity determining region (VH CDR1, VH CDR2, and VH CDR3) of the heavy chain variable region and each complementarity determining region (VL CDR1, VL CDR2, and VL CDR3) of the light chain variable region are
(A)
(a) VH CDR1: amino acid sequence shown in SEQ ID NO: 1;
(b) VH CDR2: amino acid sequence set forth in SEQ ID NO:2;
(c) VH CDR3: amino acid sequence shown in SEQ ID NO:3;
(d) VL CDR1: amino acid sequence shown in SEQ ID NO:5;
(e) VL CDR2: amino acid sequence shown in SEQ ID NO:6;
(f) VL CDR3: amino acid sequence shown in SEQ ID NO:7; or (B)
(a) VH CDR1: amino acid sequence shown in SEQ ID NO:9;
(b) VH CDR2: amino acid sequence set forth in SEQ ID NO:10;
(c) VH CDR3: amino acid sequence shown in SEQ ID NO: 11;
(d) VL CDR1: amino acid sequence shown in SEQ ID NO: 13;
(e) VL CDR2: amino acid sequence shown in SEQ ID NO: 14;
(f) VL CDR3: amino acid sequence set forth in SEQ ID NO: 15;
is an antibody or antigen-binding fragment thereof, comprising
A monoclonal antibody or antigen-binding fragment thereof that binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs. The amino acid sequences of the heavy and light chain variable regions, respectively, are:
the amino acid sequence of SEQ ID NO:4 and the amino acid sequence of SEQ ID NO:8; or the amino acid sequence of SEQ ID NO:12 and the amino acid sequence of SEQ ID NO:16;
The monoclonal antibody or antigen-binding fragment thereof according to claim 1. 3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said epitope is that of glyceraldehyde-derived AGEs. 4. The monoclonal antibody of any one of claims 1-3, which is a full length antibody, Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, or sc(Fv) ₂ or an antigen-binding fragment thereof. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a mouse antibody, humanized antibody , chimeric antibody, or antigen-binding fragment thereof. The monoclonal antibody or antigen binding thereof according to any one of claims 1 to 5, which binds to an epitope of glyceraldehyde-derived AGEs or glycolaldehyde-derived AGEs with a dissociation constant (Kd value) of 1 × 10 ^-5 M or less. piece. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6. Diabetes, impaired glucose tolerance, retinopathy, nephropathy, peripheral neuropathy, leg gangrene, arteriosclerosis, thrombosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cancer, polycystic ovary syndrome, ovarian function 8. The pharmaceutical composition according to claim 7, for use in diagnosing, treating or preventing a disease selected from disorders, central nervous system disorders and Alzheimer's disease. 9. The pharmaceutical composition according to claim 8, wherein the disease is cancer selected from melanoma, lung cancer and liver cancer. A nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6. An expression vector comprising the nucleic acid of claim 10. A host cell comprising the expression vector of claim 11.

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