Humanized monoclonal advanced glycation end product antibodies

Abstract

This invention provides a humanized monoclonal antibody that binds to advanced glycation end product (AGE) modified proteins or peptides on a cellular surface. [Solution] A humanized monoclonal antibody for advanced glycation end products, comprising at least one amino acid sequence selected from a group consisting of eight specific amino acid sequences, each with a different sequence. The antibody may be bound to a carboxymethyllysine-modified protein or peptide on a cell. The antibody can be used in a cell separation process, for example, in magnetic cell separation.

Description

[Technical Field]

[0001] [Background technology] Advanced glycation end products (AGEs), also known as AGE-modified proteins or final glycation end products, are produced from non-enzymatic reactions between sugars and protein side chains (AndoK, et al., Membrane Proteins of Human Erythrocytes Are Modified by Advanced Glycation End Products During Aging in the Circulation, Biochemical and Biophysical). Research Communications, Vol. 258, 123, 125 (1999). This process involves reducing sugars and The reaction begins with a reversible reaction with an amino group to form a Schiff base, which then forms a covalently bonded Amadori rearrangement product. Once formed, the Amadori product undergoes further rearrangement to produce an AGE.

[0002] Antibodies that bind to AGE-modified proteins on cells are known in the art. Examples include U.S. Patent No. 5,702,704 by Bucala and Al-Abed et al. Examples include antibodies described in U.S. Patent No. 6,380,165. Non-human anti-AGE antibodies are also commercially available. For example, R&D Systems, Inc. (Minneapolis, MN) sells a mouse anti-AGE antibody produced against carboxymethyllysine conjugated with keyhole limpet hemocyanin. Commercially available antibodies are designed for research or diagnostic use and may contain substances unsuitable for in vivo use in animals or humans. Such antibodies are not therapeutic antibodies and are not intended for administration to human subjects.

[0003] AGEs and AGE-modified cells are associated with several pathological conditions, including diabetic complications, inflammation, retinopathy, nephropathy, stroke, endothelial cell dysfunction, and neurodegenerative disorders (Bierhaus A, “AGEs and their interaction with AGE-receptors in vascular disease and diabetesmellitus. I. The AGE concept,” CardiovascRes, Vol. 37(3), 586-600 (1998)). AG The association between E and various pathological conditions, diseases, and disorders has led to the identification of AGEs as therapeutic targets. Therapies for targeting and eliminating AGE-modified cells include the application of ultrasound and the administration of antibodies, including humanized antibodies that bind to AGEs (see, for example, International Publication No. 2009 / 143411, U.S. Patent Application Publication No. 2013 / 0243785, and U.S. Patent Application Publication No. 2016 / 0215043). Antibody-based immunotherapy is particularly desirable due to its ability to specifically target and kill cells that express antigens to which antibodies bind, while preserving cells that do not express the antigen.

[0004] Antibodies are Y-shaped proteins composed of two heavy chains and two light chains. The two arms of the Y-shape form the antigen-binding fragment (Fab) region of the antibody, while the base or tail of the Y-shape forms the crystallizable fragment (Fc) region of the antibody. Antigen binding occurs at the terminal portion of the antigen-binding fragment region (the tip of the Y-shaped arms). This occurs at a location called a paratope, which is a pair of complementarity-determining regions (CDRs, also known as complementarity-determining regions or hypervariable regions). The region differs among various antibodies and confers binding specificity to a given antibody for a given antigen. The crystallizable fragment region of an antibody can determine the outcome of antigen binding and interact with the immune system, for example, by initiating a complement cascade or antibody-dependent cell-mediated cytotoxicity (ADCC).

[0005] Therapeutic monoclonal antibodies were first produced in mice using hybridoma technology. A significant problem associated with administering mouse and other unmodified non-human antibodies to human subjects is the risk of the human immune system attacking the non-human antibodies. Many human patients receiving mouse antibodies experience a human anti-mouse antibody response (HAMA response). This can cause an allergic reaction known as HAMA. The HAMA response can be mild, such as a rash, or severe, such as kidney failure. In addition, the human immune system often neutralizes mouse antibodies, reducing their half-life and impairing their ability to target the target antigen.

[0006] Non-human antibodies can be produced with low immunogenicity against humans by modifying antibodies to include a combination of non-human and human antibody components. Non-human antibodies are selected based on their specificity to the desired target antigen. Chimeric antibodies can be produced by combining the variable region of a non-human antibody with the constant region of a human antibody. Chimeric antibodies are approximately 70% human and are less immunogenic than unmodified non-human antibodies. Humanized antibodies can be produced by substituting the complementarity-determining region (CDR) of a human antibody with the complementarity-determining region of a non-human antibody. Humanized antibodies are approximately 95% human and contain a larger amount of human antibody components, making them less immunogenic than chimeric antibodies. Humanization is a well-known scientific technique (see, for example, U.S. Patent No. 5,693,762), and the technology has advanced to the point where custom antibody humanization services are commercially available. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent No. 5,702,704 [Patent Document 2] U.S. Patent No. 6,380,165 [Patent Document 3] International Publication No. WO 2009 / 143411 Pamphlet [Patent Document 4] U.S. Patent Application Publication No. 2013 / 0243785 [Patent Document 5] U.S. Patent Application Publication No. 2016 / 0215043 [Patent Document 6] U.S. Patent No. 5,693,762 [Non-Patent Document]

[0008] [Non-Patent Document 1] Ando K, et al., Membrane Proteins of Human Erythrocytes Are Modified by Advanced Glycation End Products During Aging in the Circulation, Biochemical and Biophysical Research Communications, Vol. 258, 123, 125 (1999) [Non-Patent Document 2] Bierhaus A, “AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept,” Cardiovasc Res, Vol. 37(3), 586 - 600 (1998) [Summary of the Invention] [Means for Solving the Problems]

[0009] In a first aspect, the present invention is a humanized monoclonal advanced glycation end product antibody comprising a heavy chain and a light chain. The heavy chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95%, and more preferably at least 98%, with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, and SEQ ID NOs: 5. The light chain comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95%, and more preferably at least 98%, with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, and SEQ ID NOs: 15. The antibody binds to a carboxymethyllysine-modified protein or peptide.

[0010] In a second aspect, the present invention is a humanized monoclonal advanced glycation end product antibody comprising a heavy chain having a heavy chain variable region and a light chain having a light chain variable region. The heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9 and SEQ ID NOs: 10. The light chain variable region comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, and more preferably at least 98% sequence identity with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 17, SEQ ID NOs: 18, SEQ ID NOs: 19 and SEQ ID NOs: 20. The antibody comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95%, and more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the above. The antibody binds to a carboxymethyl lysine-modified protein or peptide.

[0011] In a third aspect, the present invention is a humanized monoclonal advanced glycation end product antibody comprising a heavy chain and a light chain. The heavy chain comprises an amino acid sequence having at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, and 5. The light chain comprises an amino acid sequence having at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, and 15. The antibody binds to a carboxymethyllysine-modified protein or peptide.

[0012] In a fourth aspect, the present invention is a humanized monoclonal advanced glycation end product antibody comprising a heavy chain having a heavy chain variable region and a light chain having a light chain variable region. The heavy chain variable region comprises an amino acid sequence having at least one amino acid sequence selected from the group consisting of SEQ ID NOs. 7, SEQ ID NOs. 8, SEQ ID NOs. 9, and SEQ ID NOs. 10. The light chain variable region comprises an amino acid sequence having at least one amino acid sequence selected from the group consisting of SEQ ID NOs. 17, SEQ ID NOs. 18, SEQ ID NOs. 19, and SEQ ID NOs. 20. The antibody binds to a carboxymethyllysine-modified protein or peptide.

[0013] In a fifth aspect, the present invention is a composition comprising a humanized monoclonal advanced glycation end product antibody and a pharmaceutically acceptable carrier.

[0014] In a sixth aspect, the present invention relates to a method for treating a human subject diagnosed with an AGE or AGE-modified cell-related condition, disease, or disorder, comprising the step of administering a composition comprising a humanized monoclonal advanced glycation end product antibody to the subject. The antibody comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity, preferably at least 95%, and more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, and SEQ ID NOs: 5. The antibody comprises a light chain comprising an amino acid sequence having at least 90% sequence identity, preferably at least 95%, and more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, and SEQ ID NOs: 15.

[0015] definition The term "peptide" refers to a molecule consisting of 2 to 50 amino acids.

[0016] The term "protein" refers to a molecule consisting of more than 50 amino acids.

[0017] The terms “Advanced Glycation End Products,” “AGE,” “AGE-Modified Proteins or Peptides,” “Advanced Glycation Terminal Products,” and “AGE Antigens” refer to modified proteins or peptides formed as a result of a reaction between sugars and protein side chains, which then undergo further rearrangement to form irreversible crosslinks. This process begins with a reversible reaction between a reducing sugar and an amino group to form a Schiff base, which then forms a covalently bonded Amadori rearrangement product. Once formed, the Amadori product undergoes further rearrangement to produce AGEs. AGE-modified proteins and antibodies against AGE-modified proteins are described in U.S. Patent No. 5,702,704 by Bucala and U.S. Patent No. 6,380,165 by Al-Abed et al. Glycated proteins or peptides that have not undergone the necessary transformation to form AGEs, such as N-deoxyfructosyl lysine found in glycated albumin, are not AGEs. AGEs are AGE modifications (also called AGE epitopes or AGE moieties), e.g., 2-(2-flo (Iyl)-4(5)-(2-Furanyl)-1H-imidazole ("FFI"); 5-Hydroxymethyl-1-alkylpyrrole-2-carbaldehyde ("Pyrraline") ); it can be identified by the presence of the non-fluorescent model AGE 1-alkyl-2-formyl-3,4-diglicosylpyrrole ("AFGP"); carboxymethyl lysine; carboxyethyl lysine; and pentosidine. Another AGE, ALI, is described in U.S. Patent No. 6,380,165.

[0018] The terms "Advanced Glycation End Product (AGE) Antibody," "Antibody that Binds to AGE-Modified Proteins on Cells," "Anti-AGE Antibody," or "AGE Antibody" refer to antibodies that bind to AGE-modified proteins or peptides, where AGE-modified proteins or peptides are typically found bound to the cell surface. "Advanced Glycation End Product (AGE) Antibody," "Antibody that Binds to AGE-Modified Proteins on Cells," "Anti-AGE Antibody," or "AGE Antibody" do not include antibodies or other proteins that bind to both AGE-modified proteins or peptides and the same non-AGE-modified proteins or peptides with the same specificity and selectivity (i.e., binding is not increased by the presence of AGE modification). Since albumin is not typically found bound to the cell surface, AGE-modified albumin is not an AGE-modified protein on the cell surface. "Advanced Glycation End Product (AGE) Antibody," "Antibody that Binds to AGE-Modified Proteins on Cells," "Anti-AGE Antibody," or "AGE Antibody" include only antibodies that result in the removal, destruction, or death of cells. This also includes antibodies conjugated to, for example, toxins, drugs, or other chemical substances or particles.

[0019] The term "humanized antibody" refers to a genetically modified antibody in which the complementarity-determining region (CDR) of a human antibody is replaced with the CDR of a non-human antibody, resulting in an antibody variable region amino acid sequence that is closer to that of humans than that of other species.

[0020] The term "mutant" refers to a nucleotide, protein, or amino acid sequence that differs from a specifically identified sequence by the deletion, substitution, or addition of one or more nucleotides, protein, or amino acid residues. A mutant may be a spontaneously occurring allelic mutant or a non-spontaneously occurring mutant. A mutant of an identified sequence may retain some or all of the functional characteristics of the identified sequence.

[0021] The term "percent (%) sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after, if necessary, the sequence has been aligned and gaps introduced to achieve maximum percentage sequence identity, and no conservative substitutions are considered as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Preferably, the % sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is publicly available from Genentech, Inc. (South San Francisco, CA) or is stored in the user documentation of the U.S. Copyright Office, and is registered under U.S. Copyright Registration Number It is possible to compile from the source code registered in TXU510087. The ALIGN-2 program runs on UNIX operating systems, including Digital UNIX V4.0D. It should be compiled for use with ALIGN-2 Pro Set by grams and do not change.

[0022] When using ALIGN-2 for amino acid sequence comparison, the given amino acid sequence A is compared to the given amino acid sequence A. The % sequence identity to amino acid sequence B (or can be expressed as a given amino acid sequence A that has or contains a specific % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y (wherein X is the sequence alignment) The ALIGN-2 program ensures a perfect match in the program alignment of A and B. This is the number of scored amino acid residues, where Y is the total number of amino acid residues in B). If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are ALIGN-2 Comp It can be obtained using a computer program. [Brief explanation of the drawing]

[0023] [Figure 1] This graph shows the antibody binding of commercially available mouse anti-AGE antibodies. [Figure 2] This is a chromatogram of a transfected mouse monoclonal anti-AGE antibody. [Figure 3] This is a gel electrophoresis image of transfected mouse monoclonal anti-AGE antibody. [Figure 4] This graph shows the binding of transfected mouse monoclonal anti-AGE antibody to CML-OVA in an enzyme-linked immunosorbent assay. [Modes for carrying out the invention]

[0024] The present invention relates to a novel humanized monoclonal antibody that conjugates to AGE-modified proteins or peptides on a cellular basis. More specifically, the anti-AGE antibody conjugates to carboxymethyllysine-modified proteins or peptides on a cellular basis. The antibody is suitable for in vivo administration to human subjects and is preferably substantially non-immunogenic to humans. The antibody may be conjugated with a toxin or other substance for inducing cell death. The antibody may also be included in a composition together with a pharmaceutically acceptable carrier. The antibody is considered to have superior antigen-binding properties compared to comparable commercially available non-human anti-AGE antibodies.

[0025] The humanized monoclonal advanced glycation end product antibody comprises a heavy chain having a protein sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and a light chain having a protein sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. The variable domain of the humanized heavy chain may have a protein sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. The variable domain of the humanized light chain may have a protein sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.

[0026] The anti-AGE antibody binds to a protein or peptide having a carboxymethyllysine AGE modification. Carboxymethyllysine (N(epsilon)-(carboxymethyl)lysine, N(6)-carboxymethyllysine, 2-amino-6-(carboxymethylamino)hexanoic acid and also known as CML) is found in proteins or peptides and lipids as a result of oxidative stress and chemical glycation. Carboxymethyllysine-modified proteins or peptides are recognized by RAGE, a receptor expressed on a variety of cells. Carboxymethyllysine has been well studied and carboxymethyllysine-related preparations are commercially available. For example, Cell Biolabs, Inc. sells CML-BSA antigen, CML polyclonal antibody, CML immunoblot kit, and CML competitive ELISA kit (www.cellbiolabs.com / cml-assays). CML ovalbumin (CML-OVA) is a preferred control for verification of antibody binding.

[0027] The anti-AGE antibody has a low dissociation rate from the antibody-antigen complex, i.e., k d (k back or also called off-rate) and is preferably at most 6×10 -3 , 5×10 -3 , 1×10 -3 , 8×10 -4 , 5×10 -4 , 1×10 -4 , 8×10 -5 , 5×10-5 or 1 × 10 -5 (seconds -1 Preferably, the binding characteristics of the anti-AGE antibody are as shown in Figure 1, available from R&D Systems, Inc. (Minneapolis, MN; catalog number MAB3247). It is superior to Uss's carboxymethyl lysine monoclonal antibody (clone 318003).

[0028] The binding of humanized antibodies can be evaluated, for example, by concentration-dependent binding ELISA or cell-based binding assays. Preferably, the binding of humanized anti-AGE antibodies is equivalent to or better than the binding of non-human anti-AGE antibodies.

[0029] Anti-AGE antibodies can destroy AGE-modified cells through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells to which specific antibodies have bound antigens on their membrane surface. ADCC can be mediated by natural killer (NK) cells, macrophages, neutrophils, or eosinophils. Effector cells bind to the Fc site of the antibody they bind to. NK cells, for example, NK92 cells (a cell line available from NantKwest, CulverCity, CA), can be used to lyse AGE cells. When administered simultaneously with or subsequently to antibodies, complement activity is enhanced, thereby increasing the cytotoxic effect of anti-AGE antibodies. Anti-AGE antibodies also destroy AGE-modified cells through complement-dependent cytotoxicity (CDC). According to the CDC, the complement cascade of the immune system is triggered by antibodies binding to target antigens.

[0030] Anti-AGE antibodies may be coupled with substances that cause the destruction of AGE-modified cells. Examples of such substances include toxins, cytotoxic substances, magnetic nanoparticles, and magnetic spin-vortex disks.

[0031] Toxins such as pore-forming toxins (PFTs) coupled with anti-AGE antibodies (Aroian R. et al., “Pore-Forming Toxins and Cellular Non-Immune Defenses (CNIDs),” Current Opinionin Microbiology, 10:57-61 (2007)) are injected into patients to selectively target AGE-modified cells. The cells can then be removed. Anti-AGE antibodies recognize and bind to AGE-modified cells. Subsequently, the toxins cause pore formation on the cell surface, after which the cells are removed by osmotic lysis.

[0032] By injecting magnetic nanoparticles coupled with anti-AGE antibodies into patients, AGE-modified cells can be targeted and removed. By applying a magnetic field, the magnetic nanoparticles can be heated, allowing for the selective removal of AGE-modified cells.

[0033] Alternatively, a magnetic spin vortex disk, which is magnetized only when a magnetic field is applied to avoid self-aggregation that could block blood vessels, begins to rotate when a magnetic field is applied, causing membrane disruption of target cells. A magnetic spin vortex disk coupled with an anti-AGE antibody specifically targets AGE-modified cell types without eliminating other cells.

[0034] Humanized monoclonal anti-AGE antibodies or their variants may contain a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, including its post-translational modifications. A heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-AGE antibody containing such a sequence retains its ability to bind to AGE. Substitutions, insertions, or deletions may occur in any part of the sequence.

[0035] Humanized monoclonal anti-AGE antibodies or their variants include at least 90 amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, including their post-translational modifications. The heavy chain variable region may contain %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. A variable region with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-AGE antibody containing such a sequence will retain its ability to bind to AGE. Substitutions, insertions, or deletions may occur in any part of the sequence.

[0036] Humanized monoclonal anti-AGE antibodies or their variants may contain a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, including its post-translational modifications. A light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-AGE antibody containing such a sequence retains its ability to bind to AGE. Substitutions, insertions, or deletions may occur in any part of the sequence.

[0037] Humanized monoclonal anti-AGE antibodies or their variants may contain a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, including its post-translational modifications. The variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-AGE antibody containing such a sequence will retain its ability to bind to AGE. Substitutions, insertions, or deletions may occur in any part of the sequence.

[0038] Antibody fragments can be used in place of the whole antibody. Preferably, the fragments are derived from an antibody comprising a heavy chain having a protein sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, and 5, and a light chain having a protein sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, and 15. The antibody is broken down into smaller fragments by enzymatic degradation. Papain degradation cleaves the N-terminal side of the inter-heavy-chain disulfide bridge to produce a Fab fragment. The Fab fragment contains the light chain and one of the two N-terminal domains of the heavy chain (also known as the Fd fragment). Pepsin degradation cleaves the C-terminal side of the inter-heavy-chain disulfide bridge to produce an F(ab')2 fragment. The F(ab')2 fragment contains both the light chain and the two N-terminal domains linked by the disulfide bridge. Pepsin degradation produces Fv (fragment variable) and Fc( Crystallizable fragments may also be formed. The Fv fragment contains two N-terminal variable domains. The Fc fragment contains a domain that interacts with immunoglobulin receptors on cells and with the initiators of the complement cascade. Pepsin introduces a third constant domain (C) of the heavy chain. H Before step 3), immunoglobulin G may be cleaved again, generating a large fragment F(abc) and a small fragment pFc'. Alternatively, the antibody fragments can be produced by recombination.

[0039] Humanized antibody sequences can be used to predict their effectiveness by comparing them to known antibody sequences. For example, humanized antibody sequences can be analyzed visually and / or by computer modeling to identify sequences that are most likely to retain antigen binding. Humanized antibody sequences can also be screened for the presence of sequences known to increase the likelihood of an immunogenic response. For example, the presentation of a peptide sequence in the groove of an MHC class II molecule can lead to CD8 + T cell activation and immunogenic response occur. To reduce this response, antibodies can be designed to avoid incorporating "T cell epitopes" that can activate T cells, thereby reducing their binding affinity to MHC class II molecules. (Germ cell lineage mutation) is performed by mutating residues within the human framework or CDR to equivalent human germline cells. The process known as germlining can remove potential MHC-II epitopes.

[0040] Anti-AGE antibodies can be obtained by humanizing mouse monoclonal anti-AGE antibodies. The mouse monoclonal anti-AGE antibody has the heavy chain protein sequence shown in SEQ ID NO: 1 (the protein sequence of the variable domain is shown in SEQ ID NO: 6) and the light chain protein sequence shown in SEQ ID NO: 11 (the protein sequence of the variable domain is shown in SEQ ID NO: 16). The antibody is recombinantly synthesized in Chinese hamster ovary (CHO) cells. Humanized monoclonal antibodies can be produced from the above. Humanized monoclonal antibodies can be purified after synthesis. For example, antibodies can be purified using MabSelect SuReProteinA medium (GE Healthcare). It can be manufactured.

[0041] Humanized monoclonal anti-AGE antibodies can be included in the composition together with a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, isotonic and absorption retarder, etc., suitable for drug administration. Preferred examples of such carriers or diluents include water, saline solution, Ringer's solution, and glucose solution. Supplemental active compounds can also be incorporated into the composition. Solutions and suspensions used for parenteral administration may include sterile diluents such as water for injection, saline solution, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetic acid, citrate, or phosphoric acid; and osmotic regulators such as sodium chloride or glucose. pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0042] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions for the immediate preparation of sterile injection solutions or dispersions. Antibody pharmaceutical compositions suitable for injection may contain various excipients. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be liquid for administration using a syringe. Such compositions must be stable during manufacture and storage and must be stored in a manner that avoids contamination by microorganisms such as bacteria and fungi. Various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can be included to prevent microbial contamination. Isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, can be included in the composition. Compositions that can delay absorption include substances such as aluminum monostearate and gelatin. A sterile injection solution can be prepared by incorporating the required amount of antibody and any other therapeutic component into a suitable solvent, along with one or a combination of components as needed, and then sterilizing it. A method for preparing a sterile solid for the preparation of a sterile injection solution includes obtaining a solid by vacuum drying and freeze-drying.

[0043] For inhalation administration, antibodies can be delivered as an aerosol spray using a suitable nebulizer or pressurized container containing a gas such as carbon dioxide. Antibodies can also be delivered by inhalation as a dry powder using, for example, the iSPERSE® inhalation drug delivery platform (PULMATRIX, Lexington, MA).

[0044] The appropriate dose level for each type of antibody is generally about 0.01 to 500 mg per kg of patient body weight. The dose level is preferably about 0.1 to about 250 mg / kg, more preferably about 0.5 to about 100 mg / kg. The appropriate dose level may be about 0.01 to 250 mg / kg, about 0.05 to 100 mg / kg, or about 0.1 to 50 mg / kg. Within this range, the dose may be 0.05-0.5, 0.5-5, or 5-50 mg / kg. The antibody can be administered once or twice daily, for example, based on a regimen of once to four times daily. Antibodies typically have a long in vivo half-life, which allows the administration regimen to be reduced to once daily, once weekly, once every two or three weeks, once a month, or once every 60-90 days.

[0045] Patients receiving anti-AGE antibodies can be tested to determine whether the antibodies effectively eliminated AGE-modified cells. The presence of AGE-modified cells can be detected by markers associated with AGE modification, such as p16. INK4a This can be determined by measuring [the relevant factor]. Antibody administration and subsequent tests can be repeated until the desired therapeutic outcome is achieved.

[0046] Unit dose formulations can be prepared to facilitate administration and dose uniformity. A unit dose formulation refers to a physically distinct unit appropriate as a single dose for the subject being treated, containing a therapeutically effective amount of one or more types of antibodies along with the required pharmaceutical carrier. Preferably, the unit dose formulation is contained in a sealed container and is sterilized.

[0047] Any human subject diagnosed with a condition, disease, or disorder associated with AGEs or AGE-modified cells can be treated by the methods described herein. Examples of conditions, diseases, or disorders that can be treated with humanized monoclonal anti-AGE antibodies include Alzheimer's disease, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), chronic obstructive pulmonary disease (COPD), Huntington's disease, idiopathic pulmonary fibrosis, and muscular dystrophy. Physiological conditions (including Becker, Duchenne, limb-girdle, and Yamamoto muscular dystrophy), macular degeneration, cataracts, diabetic retinopathy, Parkinson's disease, progeria (including Werner syndrome and Hutchinson-Gilford progeria), vitiligo, cystic fibrosis, atopic dermatitis, eczema, arthritis (including osteoarthritis, rheumatoid arthritis, and juvenile rheumatoid arthritis), atherosclerosis, cancer and metastatic cancer (including, for example, breast cancer, triple-negative breast cancer, lung cancer, melanoma, colon cancer, renal cell carcinoma, prostate cancer, cervical cancer, bladder cancer, rectal cancer, esophageal cancer, liver cancer, oral and pharyngeal cancer, multiple myeloma, ovarian cancer, gastric cancer, pancreatic cancer, and retinal blastoma cancer), cancer treatment-related disorders or cancer Side effects of treatment, hypertension, glaucoma, osteoporosis, sarcopenia, cachexia, stroke, myocardial infarction, atrial fibrillation, transplant rejection, type 1 diabetes, type 2 diabetes, radiation exposure, HIV treatment side effects, chemical weapons exposure, poisoning, inflammation, nephropathy, Lewy body dementia, prion diseases (including bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, scrapie, chronic wasting disease, kuru disease and fatal familial insomnia), lordokyphosis, autoimmune diseases, adipose tissue loss, dryness Examples include tinea, Crohn's disease, asthma, age-related physiological effects (including "cosmetic" effects such as wrinkles, age spots, hair loss, reduction of subcutaneous adipose tissue, and thinning of the skin), idiopathic myopathy (e.g., including idiopathic inflammatory myopathy, idiopathic inflammatory myositis, polymyositis, dermatomyositis, sporadic inclusion body myositis, and juvenile myositis), multiple sclerosis, neuromyelitis optica (NMO; Devic's disease or Devic's syndrome), epilepsy, and adrenoleukodystrophy (ALD; X-linked adrenoleukodystrophy, X-ALD, cerebral ALD, or cALD).

[0048] Particularly preferred treatment groups include those diagnosed with conditions, diseases, or disorders associated with AGEs or AGE-modified cells, but who are unable to receive conventional treatment. For example, metastatic cancer is recognized as a condition associated with AGE-modified cells. Patients with metastatic cancer may be unable to receive cancer treatments such as surgery, radiotherapy, or chemotherapy due to other diagnoses, health conditions, or complications. For example, pregnant women cannot receive radiotherapy due to the risk of harming the fetus. Elderly or frail patients, such as those experiencing cancer cachexia, are not suitable candidates for surgery because they are at risk of being unable to tolerate invasive procedures. Patients with pre-existing immune system deficiencies or chronic infections are often... Because chemotherapy drugs harm the immune system, it cannot be said that it is possible to receive chemotherapy.

[0049] Anti-AGE antibodies can be used in cell separation processes, such as magnetic cell separation. In magnetic cell separation, anti-AGE antibodies are bound to magnetic beads through a process called coating. The coated magnetic beads can then specifically bind to AGE-modified cells. The AGE-modified cells bound to the coated anti-AGE antibodies on the magnetic beads then respond to an applied magnetic field, allowing them to be separated from non-AGE-modified cells. Magnetic cell separation can be used to isolate AGE-modified cells from tissue and liquid samples. The magnetic beads may be microbeads (0.5-500 μm) or nanoparticles (5-500 nm). Anti-AGE antibodies coated on magnetic beads can also be used in isolation processes such as immunoassays and immunoprecipitation. Similarly, anti-AGE antibodies coated on magnetic beads can be used to specifically target and separate AGE-modified proteins or peptides from tissue and liquid samples.

[0050] Anti-AGE antibodies are used in cell purification processes, such as immunopanning and It can be used in immunoadsorption. The purification process is useful in isolating desired or unwanted cells from tissue cultures, cell cultures, or blood. Cell purification can be used in transplantation, e.g., bone marrow transplantation, or infusion, e.g., blood transfusion. Cell purification is particularly useful in autologous stem cell transplantation to remove metastatic malignant cells and enrich beneficial stem cells during chemotherapy. AGE-modified cells can be isolated from tissue cultures, cell cultures, or blood samples by immunopanning or immunoadsorption using anti-AGE antibodies.

[0051] The single-letter amino acid sequence corresponding to Sequence ID No. 1 is: MGWTLVFLFLLSVTAGVHSQVQLLQPGAELVKPGASVKLACKASGYLFTTYWMHWLKQRPGQGLEWIGEISPTNGRAYYNARFKSEATLTVDKSSNTAYMQLSSLTSEASAVYYCA RSFGNYEFAYWGQGTLVTVSVASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK That is the case.

[0052] The single-letter amino acid sequence corresponding to Sequence ID No. 2 is: MGWTLVFLFLLSVTAGVHSEVQLLESGAEAKKPGASVKLSCKASGYLFTTYWMHWVHQAPGQRLEWMGEISPTNGRAYYNARFKSRVTITVDKSASTAYMELSSLRSEDTAVYYCA RSFGNYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK That is the case.

[0053] The single-letter amino acid sequence corresponding to Sequence ID No. 3 is: MGWTLVFLFLLSVTAGVHSQVQLVQSGAEVKKPGASVKVSCKASGYLFTTYWMHWVRQAPGQRLEWIGEISPTNGRAYYNARFKSRVTITRDTSASTAYMELSSLRSEDTAVYYCA RSFGNYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK That is the case.

[0054] The single-letter amino acid sequence corresponding to Sequence ID No. 4 is: MGWTLVFLFLLSVTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGYLFTTYWMHWVRQAPGQGLEWMGEISPTNGRAYYNARFKSRVTITADKSTSTAYMELSSLRSEDTAVYYCA RSFGNYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK That is the case.

[0055] The single-letter amino acid sequence corresponding to Sequence ID No. 5 is: MGWTLVFLFLLSVTAGVHSQVQLVQSGAEVKKPGASVKVSCEASGYLFTTYWMHWVRQAPGQGLEWMGEISPTNGRAYYNARFKSRVTITRDTSINTAYMELSRLRSDDTAVYYCA RSFGNYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK That is the case.

[0056] The single-letter amino acid sequence corresponding to sequence number 6 is: QVQLLQPGAELVKPGASVKLACKASGYLFTTYWMHWLKQRPGQGLEWIGEISPTNGRAYYNARFKSEATLTVDKSSNTAYMQLSSLTSEASAVYYCARSFGNYEFAYWGQGTLVTVSV That is the case.

[0057] The single-letter amino acid sequence corresponding to Sequence ID No. 7 is: EVQLLESGAEAKKPGASVKLSCKASGYLFTTYWMHWVHQAPGQRLEWMGEISPTNGRAYYNARFKSRVTITVDKSASTAYMELSSLRSEDTAVYYCARSFGNYEFAYWGQGTLVTVSS That is the case.

[0058] The single-letter amino acid sequence corresponding to sequence number 8 is: QVQLVQSGAEVKKPGASVKVSCKASGYLFTTYWMHWVRQAPGQRLEWIGEISPTNGRAYYNARFKSRVTITRDTSASTAYMELSSLRSEDTAVYYCARSFGNYEFAYWGQGTLVTVSS That is the case.

[0059] The single-letter amino acid sequence corresponding to sequence number 9 is: QVQLVQSGAEVKKPGSSVKVSCKASGYLFTTYWMHWVRQAPGQGLEWMGEISPTNGRAYYNARFKSRVTITADKSTSTAYMELSSLRSEDTAVYYCARSFGNYEFAYWGQGTLVTVSS That is the case.

[0060] The single-letter amino acid sequence corresponding to sequence number 10 is: QVQLVQSGAEVKKPGASVKVSCEASGYLFTTYWMHWVRQAPGQGLEWMGEISPTNGRAYYNARFKSRVTITRDTSINTAYMELSRLRSDDTAVYYCARSFGNYEFAYWGQGTLVTVSS That is the case.

[0061] The single-letter amino acid sequence corresponding to sequence number 11 is: MVSSAQFLGLLLLCFQGTRCDVVMTQTPLSLPVSLGDQASISCRSRQSLVNSNGNTFLQWYLQKPGQSPKLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHV PPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC That is the case.

[0062] The single-letter amino acid sequence corresponding to sequence number 12 is: MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLPVTLGQPASISCRSRQSLVNSNGNTFLQWLQQRPGQPPRLLIYKVSLRFSGVPDRFSGSGAGTDFTLTISRVEAEDVGIYFCSQSTHV PPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC That is the case.

[0063] The single-letter amino acid sequence corresponding to sequence number 13 is: MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLSVTPGQPASISCRSRQSLVNSNGNTFLQWYLQKPGQSPQLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYYCSQSTHV PPTFGGGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC That is the case.

[0064] The single-letter amino acid sequence corresponding to SEQ ID NO: 14 is: MVSSAQFLGLLLLCFQGTRCDVVMTQSPLSLPVTLGQPASISCRSRQSLVNSNGNTFLQWFQQRPGQSPRRLIYKVSLRFSGVPDRFSGSGSDTDFTLRISRVEAEDVGLYYCSQSTHV PPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC That is the case.

[0065] The single-letter amino acid sequence corresponding to sequence number 15 is: MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLSVTPGQPASISCRSRQSLVNSNGNTFLQWLLQKPGQPPQLLIYKVSLRFSGVPNRFSGSGSGTDFTLKISRVEAEDVGLYYCSQSTHV PPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC That is the case.

[0066] The single-letter amino acid sequence corresponding to sequence number 16 is: DVVMTQTPLSLPVSLGDQASISCRSRQSLVNSNGNTFLQWYLQKPGQSPKLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPPTFGGGTKLEIK That is the case.

[0067] The single-letter amino acid sequence corresponding to sequence number 17 is: DIVMTQTPLSLPVTLGQPASISCRSRQSLVNSNGNTFLQWLQQRPGQPPRLLIYKVSLRFSGVPDRFSGSGAGTDFTLTISRVEAEDVGIYFCSQSTHVPPTFGQGTKVEIK That is the case.

[0068] The single-letter amino acid sequence corresponding to sequence number 18 is: DIVMTQTPLSLSVTPGQPASISCRSRQSLVNSNGNTFLQWYLQKPGQSPQLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCSQSTHVPPTFGGGTKVEVK That is the case.

[0069] The single-letter amino acid sequence corresponding to sequence number 19 is: DVVMTQSPLSLPVTLGQPASISCRSRQSLVNSNGNTFLQWFQQRPGQSPRRLIYKVSLRFSGVPDRFSGSGSDTDFTLRISRVEAEDVGLYYCSQSTHVPPTFGQGTKLEIK That is the case.

[0070] The single-letter amino acid sequence corresponding to sequence number 20 is: DIVMTQTPLSLSVTPGQPASISCRSRQSLVNSNGNTFLQWLLQKPGQPPQLLIYKVSLRFSGVPNRFSGSGSGTDFTLKISRVEAEDVGLYYCSQSTHVPPTFGGGTKVEIK That is the case. [Examples] [Examples]

[0071] Affinity and kinetics of commercially available anti-AGE antibodies The affinity and kinetics of commercially available mouse anti-AGE product antibodies were tested. Anti-AGE antibodies produced against carboxymethyllysine coupled to keyhole limpet hemocyanin (clone 318003) were obtained (R&D Systems, Inc., Minneapolis, MN; catalog number MAB3247). Nα,Nα-bis(carboxymethyl)-L-lysine trifluoroacetate (Sigma-Aldrich, St. Louis, MO) was used as a model substrate for AGE-modified proteins in cells. Label-free interaction analysis was performed on a BIACORE® T200 (GE Healthcare, Pittsburgh, PA) using a Series S sensor chip CM5 (GE Healthcare, Pittsburgh, PA), with Fc1 set as a blank and Fc2 fixed with the test antibody (molecular weight 150,000 Da). The electrophoresis buffer was HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% P-20, pH 7.4) at 25°C. The software used was BIACORE® T200 evaluation software. The result was version 2.0. A dual control (injection of Fc2-1 and buffer solution only) was used in the analysis, and the data were fitted to a Langmuir 1:1 coupled model.

[0072] [Table 1]

[0073] Figure 1 shows a graph of the antibody response against time. The following values ​​were determined by analysis: k a (1 / Ms) = 1.857 × 10 3 , k d (1 / sec)=6.781×10 -3 , K D (M) = 3.651 × 10 -6 , R max (RU)=19.52 and Kai 2 =0.114. Fitting chi 2 The value is R max Since it is less than 10%, the goodness of fit is Trustworthy. [Examples]

[0074] Transient expression of mouse monoclonal anti-AGE antibody Mouse monoclonal anti-AGE antibodies were transfected into Chinese hamster ovary (CHO) cells, and sufficient amounts of antibody were expressed and purified for evaluation by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis. The DNA encoding the antibody's amino acid sequence was synthesized. This DNA was cloned into the mammalian transient expression plasmid pD2610-v13 (DNA2.0).

[0075] CHO cells adapted to suspension (Thermo Fisher, UK) 2.0-3.0 × 10⁻¹⁶ 5 Thin Cells / mL in a 500mL Erlenmeyer flask with a vent (Corning, Netherlands) ) containing 8 mM L-glutamine (Thermo Fisher, UK) and 10 mL / L hypo ProCHO-4 serum-free medium (Lonza) supplemented with xanthine / thymidine (Thermo Fisher, UK) The cells were cultured at 135 rpm, 8% CO2, and 37°C in a container (ThermoFisher, Belgium). Maxiprep constructs were prepared using the PureLink® HiPure plasmid filter Maxiprep kit (ThermoFisher, UK). Vector DNA was measured using a NanoDrop Lite spectrophotometer. The quantity was determined using [a specific method / tool].

[0076] Final density 1.0×10 6 500 mL of cells / mL were transiently transfected with 1.25 μg / mL vector DNA, and 8 mM L-glutamine (Invitrogen, UK) was added in a 500 mL Erlenmeyer flask with aeration holes (Corning, Netherlands). The cultures were incubated in ProCHO-5 serum-free medium (Lonza, Belgium) supplemented with 10 mL / L hypoxanthine / thymidine (Invitrogen, UK). The cultures were incubated at 37°C in 8% CO2 and 13 Incubate at 5 rpm for 8 days and feed with 7.5% (v / v) Power Feed A (Sartorius). The sample (from Germany) was administered daily every 2-3 days, and then recovered by centrifugation at 4000 rpm and 4°C for 40 minutes. 612 mL of antibody was prepared by transfection.

[0077] Using an AKTA chromatography system (GE Healthcare) at room temperature (19°C), antibody chromatography The preparation was carried out. After centrifugation, filtered (0.22 μm) cell culture supernatant was applied to an AKTA system equipped with a 1 mL HiTrap Protein A column equilibrated with wash buffer. After loading, the column was washed with 20 column volumes of wash buffer. The bound antibody was serially eluted with 10 column volumes of elution buffer. Figure 2 shows the antibody chromatogram at 280 nm. All eluted fractions were neutralized with Tris buffer at pH 9.0. Eluted fractions corresponding to the elution peaks were selected and dialyzed overnight in PBS at 4°C.

[0078] The purity of the antibody was evaluated using sodium dodecyl sulfate / polyacrylamide gel electrophoresis (SDS-PAGE). The antibody was found to be over 95% pure. Figure 3 shows the gel electrophoresis of the antibody. Under reducing conditions, both the heavy and light chains of the antibody were visible to the naked eye and were observed at approximately 50 and 25 kDa, respectively, of the expected molecular weight. Under non-reducing conditions, a single major band was observed. The absence of any additional major bands indicates the absence of antibody aggregates.

[0079] The purified antibody concentrate was evaluated by spectrophotometric analysis. Using a NanoDrop Lite spectrophotometer, the extinction coefficient of 205,500 M⁻¹·cm⁻¹ (or A280 with 1.0 mg / mL = 1.37 [assuming MW = 150,000 Da]) was used as the standard reference for IgG in A280. The antibody was quantified according to the instructions of the expert. 600 mL of transfected mouse antibody at a concentration of 0.6 mg / mL was purified to 2.3 mL to obtain a total yield of 1.4 mg of antibody.

[0080] The binding of the transfected antibody was evaluated by ELISA. CML-OVA / N ε-(carboxymethyl)lysine-OVA (Circulex, Japan, catalog number CY-R2053) 100 ng / well was fixed overnight at 4°C in coating buffer (0.05 M NaHCO3 adjusted to pH 9.5 by adding 0.05 M Na2CO3) on a 96-well MaxiSorp® plate. The coating buffer was removed and the plate was washed three times with PBS Tween (PBS-T) (0.1% (v / v) Tween 20). 3% in PBS 200 μL of (w / v) skim milk was added to each well and stirred at room temperature for 2 hours. The plate was then washed three times with PBS-T.

[0081] The antibody was diluted from 1,000 ng / mL to 0.488 ng / mL in incubation buffer (PBS, 1% (w / v) BSA). 100 μL of the diluted antibody was added to each of the three wells of the plate and stirred at room temperature for 2 hours.

[0082] The wells were washed three times with PBS-T. After washing, goat anti-mouse HRP (Fc-specific) (Bio Rad, catalog number 0300) was diluted 1:5,000 in incubation buffer. -0108P) Add 100 μL per well to all wells and agitate the plate at room temperature for 1 hour. Wash the wells three times with PBS-T. After washing, add 100 μL of TMB substrate to each well and incubate at 37°C for 10 minutes. Add 50 μL of 1 M HCl to each well and immediately scan the plate at 450 nm on a Tecan Sunrise plate reader. The data was read. Figure 4 shows the ELISA results for antibody binding to CML-OVA. The values ​​shown in the graph are the average of three readings.

[0083] The ELISA results indicate that the transfected antibody recognizes and binds to the CML-OVA protein, a known AGE-modifying protein. The results confirm that the antibody sequence is correct and the antibody is active. Similar results are expected for humanized monoclonal anti-AGE antibodies containing the complementarity-determining region of such mouse antibodies. [Examples]

[0084] Production of humanized antibodies The mouse anti-AGE antibody was sequenced. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 11. The amino acid sequences of the variable domains of the heavy chain and light chain are shown in SEQ ID NO: 6 and SEQ ID NO: 16, respectively.

[0085] CDR residues in mouse heavy chains were identified using IMGT and the Kabat numbering system. We determined the human germline gene V region closest to the heavy chain variable region. We searched online databases of human IgG sequences and used the BLAST search algorithm to identify the mouse heavy chain variable region. Compared to the main domain, candidate human variable domains were selected from the top 200 results of BLAST. These were narrowed down to four candidates based on framework homology, maintenance of key framework residues, and combinations of standard loop structures.

[0086] Four humanized heavy chain variable domain (HDRs) were generated by transplanting the mouse heavy chain variable domain (HDR) into four acceptor frameworks. The amino acid sequences of the four humanized HDRs are shown in SEQ ID NOs: 7, 8, 9, and 10. The homology of the humanized HDRs was compared with that of the mouse heavy chain variable domain. The results of the homology comparison are shown in Table 2 below.

[0087] [Table 2]

[0088] In order of homology, Sequence ID No. 7 was the most similar to the mouse heavy chain variable domain, followed by Sequence ID No. 9, Sequence ID No. 8, and Sequence ID No. 10.

[0089] Mouse light chain CDR residues were identified using IMGT and the Kabat numbering system. We determined the human germline gene V region closest to the light chain variable region. We searched online databases of human IgK sequences and used the BLAST search algorithm to identify the mouse light chain variable region. Compared to the main domain, candidate human variable domains were selected from the top 200 results of BLAST. These were narrowed down to four candidates based on a combination of framework homology, maintenance of key framework residues, and limiting loop structure.

[0090] Four humanized light chain variable domain (CDRs) were created by transplanting the mouse light chain variable domain into four acceptor frameworks. The amino acid sequences of the four humanized light chain variable domains are shown in SEQ ID NOs: 17, 18, 19, and 20. The homology of the humanized light chain variable domains was compared with that of the mouse light chain variable domain. The results of the homology comparison are shown in Table 3 below.

[0091] [Table 3]

[0092] In order of homology, sequence number 18 was the most similar to the mouse light chain variable domain, followed by sequence numbers 20, 19, and 17.

[0093] Humanized heavy chain and light chain variable domain variants are examined to determine whether they are humanized according to the World Health Organization's (WHO) definition of humanized antibodies. The determination was made based on the following criteria. The WHO states that an antibody is humanized if its variable region amino acid sequence is closer to that of humans than to that of other species. Sequence IDs 7, 8, 9, 10, 17, 18, 19, and 20 were evaluated using the Immunogenetics Information System (IMGT) DomainGapAlign tool. (Ehrenmann F. et al., “IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF”, Nucleic Acids Research, Vol. 38, D301-307). All humanized variable domains. It was closer to a human than a mouse. Therefore, all humanization variable domains meet the WHO definition of a humanized antibody.

[0094] The heavy and light chain variable domains of mouse antibodies, as well as eight humanized heavy and light chain mutant sequences, were screened for MHCII-binding peptides, and an in silico algorithm was used to determine whether high-affinity peptide sequences were removed during the humanization process. Human heavy chain germline sequences IGHV1-46 and IGHV1-3, and human light chain germline sequences IGKV2-30 and IGKV2-29 were also analyzed and compared. These are typical of over 99% of the world's population and are standard alleles used to predict MHC class II epitopes. The following eight alleles, which constitute a set of genes, were screened for sequences: DRB1*01:01; DRB1*03:01; DRB1*04:01; DRB1*07:01; DRB1*08:02; DRB1*11:01; DRB1*13:02; DRB1*15:01.

[0095] The mouse heavy chain variable domain contains two high-affinity T cell epitope cores (IC). 50They have a <50nM value. Human germline sequences IGHV1-46 and SEQ ID NOs. 7, 9, and 10 each had one potential T cell epitope. Human germline sequences IGHV1-3 and SEQ ID NOs. 8 each had two potential T cell epitopes. Because human germline sequences are unlikely to be immunogenic, the potential T cell epitopes may be an overprediction by MHC class II epitope software. The potential T cell epitopes may rather be regulatory T cell epitopes, which is beneficial to the sequences.

[0096] The mouse light chain variable domain and SEQ ID NOs. 17, 18, 19, and 20 are two high-affinity T cell epitope cores (IC 50 Each had a <50nM) and one potential T cell epitope. Human germline sequence IGKV2-30 did not have a potential T cell epitope. Human germline sequence IGKV2-29 had two potential T cell epitopes. As with heavy chain variable sequences, potential T cell epitopes may be overpredictions by MHC class II epitope software and may rather be beneficial regulatory T cell epitopes.

[0097] Post-translational modifications of mouse and humanized antibodies were examined. The N-linked glycosylation motif NXS / T (where X is any amino acid other than proline) was not present in any of the variable domains. The sequences were also analyzed for the presence of amino acid motifs SNG, ENN, LNG, and LNN, which may have a tendency to deamidate asparagine to aspartic acid. The motif SNG was present in CDR1 of all light chains. Although this motif is potentially immunogenic, substitution was not performed because it occurs only in CDR.

[0098] Mouse heavy chain and light chain signal peptides were identified. These signal peptides can be expressed at high levels in Chinese hamster ovary (CHO) cells. The heavy chain signal peptide is contained in the mouse heavy chain (SEQ ID NO: 1) and four humanized heavy chains (SEQ ID NO: 2, 3, 4, and 5). The light chain signal peptide is contained in the mouse light chain (SEQ ID NO: 11) and four humanized light chains (SEQ ID NO: 12, 13, 14, and 15).

[0099] The structure of the variable domain binding site was modeled using DNASTAR NovaFold, an I-Tasser-based protein structure prediction software. NovaFold utilizes the I-Tasser algorithm, which combines threading and ab initio folding techniques to construct accurate full 3D atomic models of proteins with previously unknown structures. Analysis of the protein structures showed that the heavy chain and light chain variable domain combinations SEQ ID NOs. 7-17, 7-18, 8-20, and 9-18 are thought to have the closest structure to the mouse heavy chain and light chain variable domain combination SEQ ID NOs. 5-16. Generally, humanized mutants containing the light chain variable domain with the sequence shown in SEQ ID NO. 18 had a more favorable structure than humanized mutants containing other light chain variable domains. Similarly, humanized mutants containing the heavy chain variable domain with the sequence shown in SEQ ID NO. 7 had a more favorable structure than humanized mutants containing other heavy chain variable domains. [Examples]

[0100] (Hypothetical example): Future antibody testing Each heavy chain variable domain (SEQ ID NOs. 7, 8, 9, and 10) is synthesized in frame using the human IgG1 isotype constant domain sequence. The entire heavy chain sequence is codon-optimized (DNA2.0, USA) and validated as a DNA sequence. The amino acid sequence of the IgG1 constant domain (allotype G1m17,1) is shown below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0101] Each light chain variable domain (SEQ ID NO: 17, 18, 19, and 20) is synthesized in-frame using the human IgK isotype constant domain sequence. The entire light chain sequence is codon-optimized (DNA2.0, USA) and validated as a DNA sequence. The amino acid sequence of the IgK constant domain (allotype Km3) is shown below. TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0102] Each mutant chain is validated by DNA sequencing analysis. Subsequently, transient transfection and expression of each humanized antibody are performed. One chimeric antibody is expressed and used as a positive control, containing the mouse variable domain and the human Ig constant domain. As shown in Table 4 below, 16 humanized mutants containing humanized heavy chain and light chain variable domains and the human Ig constant domain are expressed.

[0103] [Table 4] [Examples]

[0104] (Hypothetical example): Treatment of sarcopenia Elderly patients are diagnosed with sarcopenia. A humanized monoclonal anti-AGE antibody, having a heavy chain with 99% sequence identity to SEQ ID NO: 2 and a light chain with 99% sequence identity to SEQ ID NO: 12, is administered to women. The antibody is administered intravenously at a dose of 5 mg / kg once a week. The antibody specifically targets and kills cells that express advanced glycation end products (AGEs), such as senescent cells, on their cell surface. Patient p16 levels are monitored before and after antibody administration. INK4a The effectiveness of the treatment is determined by measuring the levels. The patient does not develop an immune response to the antibodies. The patient's sarcopenia improves, as evidenced by the increase in muscle mass. [Examples]

[0105] (Hypothetical example): Treatment of osteoarthritis The patient is diagnosed with osteoarthritis. A pharmaceutically acceptable carrier, a heavy chain variable sequence with 98% sequence identity to SEQ ID NO: 7, and a light chain variable sequence with 98% sequence identity to SEQ ID NO: 18. A composition containing a humanized monoclonal anti-AGE antibody having a chain variable region is administered to males. The antibody is administered orally once daily at a dose of 10 mg / kg. The antibody specifically targets and kills cells that express advanced glycation end products, such as senescent chondrocytes, on their cell surface. The patient's p16 before and after administration of the composition. INK4a The effectiveness of the treatment is determined by measuring the levels. The patient does not develop an immune response to the antibody-containing composition. The patient's osteoarthritis improves, as evidenced by the reduction in joint pain.

[0106] References 1. International Application Pub. No. WO 2009 / 143411 to Gruber (26 Nov. 2009). 2. US Patent No. 5,702,704 to Bucala (issuedDecember 30, 1997). 3. U.S. Patent No. 6,380,165 to Al-Abed et al. (issued April 30, 2002). 4. U.S. Patent No. 6,387,373 to Wright et al. (issued May 14, 2002). 5. U.S. Patent No. 4,217,344 to Vanlerberghe et al.(issued August 12, 1980). 6. U.S. Patent No. 4,917,951 to Wallach (issued April 17, 1990). 7. U.S. Patent No. 4,911,928 to Wallach (issued March 27, 1990). 8. U.S. Patent Application Publication Pub. No. US 2010 / 226932 to Smith etal.(September 9, 2010). 9. Ando K, et al., “Membrane Proteins of HumanErythrocytes Are Modified by Advanced Glycation EndProducts During Aging inthe Circulation,” Biochemical and Biophysical ResearchCommunications,Vol. 258, 123-27 (1999). 10. Lindsey JB, et al., “Receptor ForAdvancedGlycation End-Products (RAGE) and soluble RAGE (sRAGE):Cardiovascular Implications,” Diabetes Vascular Disease Research, Vol. 6(1), 7-14, (2009). 11. Bierhaus A, “AGEs andtheirinteraction with AGE-receptors in vascular disease and diabetes mellitus.I. The AGE concept,”Cardiovasc Res, Vol. 37(3),586-600 (1998). 12. Meuter A., et al. “Markersof cellular senescence are elevated in murine blastocysts cultured in vitro:molecular consequences of culture in atmosphericoxygen” J Assist ReprodGenet. 2014 Aug 10. [Epub ahead of print]. 13. Baker, D.J. et al., “Clearance of p16Ink4a-positivesenescentcells delays ageing-associated disorders”,Nature, vol.479, pp. 232-236, (2011). 14. Jana Hadrabova, et al. “Chickenimmunoglobulins for prophylaxis: Effect of inhaled antibodies on inflammatoryparameters in rat airways” Journal of AppliedBiomedicine (in press;Available online 5 May 2014). 15. Vlassara, H. et al., “High-affinity-receptor-mediatedUptake and Degradationof Glucose-modified Proteins: A Potential Mechanism for theRemovalof Senescent Macromolecules”, Proc. Natl. Acad.Sci.USA, Vol. 82, 5588, 5591 (1985). 16. Roll, P. et al., “Anti-CD20 Therapy in Patientswith Rheumatoid Arthritis”,Arthritis & Rheumatism,Vol. 58, No. 6, 1566-1575(2008). 17. Kajstura, J. et al., “Myocite Turnover in the Aging HumanHeart”, Circ. Res., Vol.107(11), 1374-86, (2010). 18. de Groot, K. et al., “Vascular Endothelial Damageand Repair in Antineutrophil CytoplasmicAntibody-Associated Vasculitis”, Arthritis andRheumatism, Vol. 56(11), 3847, 3847 (2007). 19. Manesso, E. et al., “Dynamicsof β-CellTurnover: Evidence for β-Cell Turnover and Regenerationfrom Sources of β-Cells other than β-cell Replication in theHIP Rat”, Am. J. Physiol.Endocrinol. Metab., Vol. 297, E323, E324 (2009). 20. Kirstein, M. et al., “Receptor-specific Inductionof Insulin-like Growth Factor I in Human Monocytesby Advanced GlycosylationEnd Product-modified Protein s”, J. Clin. Invest., Vol. 90, 439, 439-440 (1992). 21. Murphy, J. F., “Trends in cancer immunotherapy”, Clinical Medical Insights: Oncology, Vol. 14(4), 67-80 (2010). 22. Virella, G. et al., “AutoimmuneResponse to Advanced Glycosylation End-Products of Human LDL”, Journal of Lipid Research, Vol. 44, 487-493 (2003). 23. Ameli, S. et al., “Effectof Immunization With Homologous LDL and Oxidized LDLonEarly Atherosclerosis in Hypercholesterolemic Rabbits”,Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 16, 1074(1996). 24. “Sarcopenia”, availableonline at en.wikipedia.org / wiki / Sarcopenia(November 14, 2014). 25. “What is sarcopenia?”,availableonline at www.iofbonehealth.org / what-sarcopenia (2014). 26. Blahd, W., “Sarcopeniawith aging”, available onlineatwww.webmd.com / healthy-aging / sarcopenia-with-aging (August 3, 2014). 27. “Keyhole limpet hemocyanin”,availableonline at en.wikipedia.org / wiki / Keyhole_limpet_hemocyanin(April18, 2014). 28. “CML-BSA Product Data Sheet”, available onlineatwww.cellbiolabs.com / sites / default / files / STA-314-cml-bsa.pdf (2010). 29. “CML (N-epsilon-(Carboxymethyl)Lysine) Assays andReagents”, available onlineatwww.cellbiolabs.com / cml-assays (Accessed on December 15, 2014). 30. Cruz-Jentoft, A. J. et al., “Sarcopenia: Europeanconsensus on definition and diagnosis”, Age and Ageing,Vol. 39, pp. 412-423 (April 13, 2010). 31. Rolland, Y. et al., “Sarcopenia: its assessment,etiology, pathogenesis, consequences and futureperspectives”, J. Nutr.Health Aging, Vol. 12(7), pp.433-450 (2008). 32. Mera, K. et al., “Anautoantibody against N ε-(carboxyethyl)lysine(CEL):Possible involvement in the removal of CEL-modified proteins bymacrophages”, Biochemical and BiophysicalResearchCommunications, Vol. 407, pp. 420-425 (March12, 2011). 33. Reddy, S. et al., “N ε -(carboxymethyl)lysine is a dominantadvancedglycation end product (AGE) antigen in tissue proteins”,Biochemistry, Vol. 34, pp. 10872-10878 (August 1, 1995). 34. Naylor, R. M. et al., “Senescent cells: a noveltherapeutic target for agingand age-related diseases”,Clinical Pharmacology &Therapeutics, Vol. 93(1), pp.105-116 (December 5,2012). 35. Katcher, H. L., “Studiesthat shed new light on aging”, Biochemistry(Moscow),Vol. 78(9), pp. 1061-1070 (2013). 36. Ahmed, E. K. et al., “Protein Modification andReplicative Senescence of WI-38 Human EmbryonicFibroblasts”, Aging Cells, Vol. 9, 252, 260 (2010). 37. Vlassara, H. et al., “AdvancedGlycosylation Endproducts on Erythrocyte CellSurfaceInduce Receptor-Mediated Phagocytosis byMacrophages”,J. Exp. Med., Vol. 166, 539, 545 (1987). 38. Fielding, R. A., et al., “Sarcopenia: anundiagnosed condition in older adults. Current consensus definition:prevalence, etiology, and consequences”, Journal of theAmerican Medical DirectorsAssociation, Vol. 12(4), pp. 249-256 (May 2011). 39. Maass, D. R. et al., “Alpaca(Lama pacos) as aconvenient source of recombinant camelid heavy chainantibodies(VHHs)”, Journal of Immunological Methods,Vol.324, No. 1-2, pp. 13-25 (July 31, 2007). 40. Strietzel, C.J. et al., “Invitrofunctional characterization of feline IgG s”, Veterinary Immunology and Immunopathology, Vol.158,pp. 214-223 (2014). 41. Patel, M. et al., “Sequence of the dogimmunoglobulin alpha and epsilon constant region genes”,Immunogenetics, Vol. 41, pp. 282-286 (1995). 42. Wagner, B. et al., “The complete map of the Igheavy chain constant gene region reveals evidence forseven IgG isotypes andfor IgDin the horse”, TheJournal ofImmunology, Vol. 173, pp. 3230-3242 (2004). 43. Hamers-Casterman, C. et al., “Naturally occurring antibodies devoid of light chains”, Nature, Vol. 363, pp. 446-448 (June 3, 1993). 44. De Genst, E. et al., “Antibodyrepertoire development incamelids”, Developmental& Comparative Immunology,Vol. 30, pp. 187-198 (available online July 11,2005). 45. Griffin, L.M. et al., “Analysis of heavy and lightchain sequences of conventional camelid antibodiesfrom Camelus dromedarius andCamelus bactrianus species ”,Journal ofImmunological Methods, Vol. 405, pp. 35-46 (available online January18, 2014). 46. Nguyen, V.K. et al., “Camel heavy-chain antibodies:diverse germline V HH andspecific mechanisms enlarge theantigen-bindingrepertoire”, The European MolecularBiologyOrganization Journal, Vol. 19, No, 5, pp. 921-930 (2000). 47. Muyldermans, S. et al., “Sequenceandstructure of V H domain from naturallyoccurring camel heavy chain immunoglobulinslacking light chains”, Protein Engineering, Vol. 7, No.9, pp. 1129-1135 (1994). 48. Glover, A., “Of mice and men”, European Biopharmaceutical Review, Winter 2016, 4 pages(January2016). 49. “The basic guide to magnetic bead cell separation”, Sepmag, available onlineatwww.sepmag.eu / free-basic-guide-magnetic-bead-cell-separation (downloadedApril12, 2017).

Claims

[Claim 1] A humanized monoclonal antibody for a cell isolation process, comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

Images