HUMANIZED MONOCLONAL ANTIBODY OF FINAL PRODUCT OF ADVANCED GLYCUS

专利摘要:
the present invention relates to a humanized monoclonal antibody that binds to a protein or peptide modified with advanced glycation end product in a cell comprising a heavy chain and a light chain. the antibody binds to a protein or peptide modified with carboxymethyl lysine. a composition comprises a humanized monoclonal antibody that binds to a protein or peptide modified with advanced glycation end product in a cell and a pharmaceutically acceptable carrier.
公开号:BR112019021471A2
申请号:R112019021471-2
申请日:2018-04-13
公开日:2020-05-12
发明作者:S. Gruber Lewis
申请人:Siwa Corporation；
IPC主号:

专利说明:

Descriptive Report on the Invention Patent for HUMANIZED MONOCLONAL ANTIBODY OF FINAL PRODUCT OF ADVANCED GLYCUS.
BACKGROUND
[0001] Advanced glycation end products (AGEs; also known as AGE-modified proteins, or end glycation products) arise from a non-enzymatic reaction of sugars with protein side chains (Ando, K. et al., Membrane Proteins of Human Erythrocytes Are Modified by Advanced Glycation End Products during Aging in the Circulation, Biochem Biophys Res Commun., Vol. 258, 123, 125 (1999)). This process begins with a reversible reaction between a reducing sugar and an amino group to form a Schiff base, which starts to form a covalently linked Amadori Rearrangement product. Once formed, Amadori product undergoes additional rearrangement to produce AGEs.
[0002] Antibodies that bind to an AGE-modified protein in a cell are known in the art. Examples include those described in U.S. 5,702,704 from Bucala and US 6,380,165 from Al-Abed et al. Non-human anti-AGE antibodies are also commercially available. For example, R&D Systems, Inc. (Minneapolis, MN) sells a murine anti-AGE antibody produced against carboxymethyl lysine conjugated to keyhole limpet hemocyanin. The commercially available antibodies are designed for laboratory or diagnostic purposes and may contain material that is not suitable for use in vivo in animals or humans. These antibodies are not therapeutic antibodies and are not intended for administration to a human subject.
[0003] AGEs and AGE modified cells have been associated with several pathological conditions, including diabetic complications, inflammation, retinopathy, nephropathy, stroke,
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2/44 endothelial cell dysfunction, and neurodegenerative disorders (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 to 600 (1998 )). The association between AGEs and various pathological conditions, diseases and disorders has led to the identification of AGEs as a therapeutic target. Therapies to target and remove AGE-modified cells include applying ultrasound and administering antibodies, including humanized antibodies, that bind to AGEs (see, for example, WO 2009/143411, US 2013/0243785 and US 2016/0215043 ). Antibody-based immunotherapies are particularly desirable because of their ability to specifically target and kill cells that express the antigen to which the antibody binds, sparing 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 form form the antigen-binding region of the fragment (Fab) while the base or tail of the Y-form forms the crystallizable fragment (Fc) region of the antibody. Antigen binding occurs at the terminal portion of the fragment's antigen binding region (the Y-shaped arms' tips) at a location known as the paratope, which is a set of complementarity determining regions (also known as CDRs or region hypervariable). The complementarity-determining regions vary between different antibodies and give a specific antibody its specificity for binding to a particular antigen. The crystallizable region of the antibody fragment determines the result of antigen binding and can interact with the immune system, such as triggering the complement cascade or initiating antibody-dependent cell-mediated cytotoxicity (ADCC).
[0005] Therapeutic monoclonal antibodies have been produced
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3/44 initially in mice using the hybridoma technique. A significant problem with the administration of murine antibodies and other unmodified non-human antibodies to human individuals is the risk of the human immune system attacking non-human antibodies. Many human patients who receive murine antibodies develop an allergic reaction called the response to human anti-mouse antibodies (HAMA response). The HAMA response may be mild, such as a life-threatening rash, such as kidney failure. In addition, the human immune system generally neutralizes murine antibodies, reducing their half-life and impairing their ability to target the intended antigen.
[0006] Non-human antibodies may be less immunogenic to humans, modifying the antibodies to contain a combination of components of non-human and human antibodies. The non-human antibody is chosen for its specificity for a desired target antigen. A chimeric antibody can be produced by combining the variable region of a non-human antibody with a human constant region. Chimeric antibodies are approximately 70% human and are less immunogenic than unmodified non-human antibodies. A humanized antibody can be produced by replacing the complementarity determining regions (CDRs) of a human antibody with those of a non-human antibody. Humanized antibodies are approximately 95% human and are less immunogenic than chimeric antibodies due to the inclusion of a greater amount of human antibody components. Humanization is a well-known scientific technique (see, for example, US 5,693,762) and has progressed to the point that personalized antibody humanization services are commercially available.
SUMMARY
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[0007] In a first aspect, the invention is a humanized monoclonal antibody of the advanced glycation end product comprising a heavy chain and a light chain. The heavy chain comprises an amino acid sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The light chain comprises an amino acid sequence with at least 90% sequence identity, preferably at least 95 % sequence identity, more preferably at least 98% sequence identity, with at least one amino acid 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 antibody binds to a protein or peptide modified with carboxymethyl lysine.
[0008] In a second aspect, the invention is a humanized monoclonal antibody of the advanced glycation end product 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, more preferably at least 98% sequence identity, with at least one amino acid 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 light chain variable region comprises an amino acid sequence having at least 90% identity of sequence, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least
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5/44 minus an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. The antibody binds to a modified protein or peptide with carboxymethyl lysine.
[0009] In a third aspect, the invention is a humanized monoclonal antibody of the advanced glycation end product 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 NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The light chain comprises an amino acid sequence having at least one amino acid 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 antibody binds to a protein or peptide modified with carboxymethyl lysine.
[0010] In a fourth aspect, the invention is a humanized monoclonal antibody of the advanced glycation end product 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 NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 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 NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. The antibody binds to a protein or peptide modified with carboxymethyl lysine.
[0011] In a fifth aspect, the invention is a composition comprising humanized monoclonal antibody of the final product of
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6/44 advanced glycation and a pharmaceutically acceptable carrier.
[0012] In a sixth aspect, the invention is a method of treating a human individual who has been diagnosed with a pathological condition, disease or disorder associated with AGEs or AGE modified cells, comprising administering to the individual a composition comprising a humanized monoclonal antibody of the final glycation end product. The antibody comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one selected amino acid sequence from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The antibody comprises a light chain comprising an amino acid sequence with at least 90% identity sequence, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 14 and SEQ ID NO: 15.
DEFINITIONS
[0013] The term peptide means a molecule composed of 2 to 50 amino acids.
[0014] The term protein means a molecule composed of more than 50 amino acids.
[0015] The terms advanced glycation end product, AGE, AGE modified protein or peptide, end glycation product and AGE antigen refer to modified proteins or peptides that are formed as a result of the reaction of sugars with protein side chains that rearrange and form cross-links
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7/44 irreversible. 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 linked Amadori rearrangement product. Once formed, Amadori's product undergoes additional rearrangement to produce AGEs. AGE-modified proteins and antibodies to AGE-modified proteins are described in U.S. 5,702,704 from Bucala and U.S. 6,380,165 from Al-Abed et al. Glycated proteins or peptides that have not undergone the rearrangement necessary to form AGEs, such as the N-deoxyfructosillisin found in glycated albumin, are not AGEs. AGEs can be identified by the presence of AGE modifications (also known as AGE epitopes or AGE moieties), such as 2- (2-furoyl) -4 (5) - (2-furanyl) -1H-imidazole (FFI); 5-hydroxymethyl-1-alkylpyrrole-2carbaldehyde (Pyrralin); 1-alkyl-2-formyl-3,4-diglycosyl pyrrole (AFGP), a non-fluorescent AGE model; carboxymethyl lysine; carboxyethyl lysine; and pentosidine. ALI, another AGE, is described in U.S. 6,380,165.
[0016] The terms antibody of the advanced glycation end product, antibody that binds to an AGE-modified protein in a cell, anti-AGE antibody or AGE antibody means an antibody that binds to an AGE-modified protein or peptide, where the AGE-modified protein or peptide is a protein or peptide normally found attached to the surface of a cell. An advanced glycation end product antibody, antibody that binds to an AGE-modified protein in a cell, anti-AGE antibody or AGE antibody does not include an antibody or other protein that binds with the same specificity and selectivity to either the protein or AGE-modified peptide for the same protein or non-AGE-modified peptide (ie, the presence of the AGE-modified does not increase binding). AGE-modified albumin is not an AGE-modified protein in a cell, because albumin is not
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8/44 a protein normally found attached to the surface of cells. An advanced glycation end product antibody, antibody that binds to an AGE-modified protein in a cell, anti-AGE antibody, or AGE antibody includes only those antibodies that lead to cell removal, destruction, or death. Also included are antibodies that are conjugated, for example, to a toxin, drug or other chemical or particle.
[0017] The term humanized antibody means a genetically modified antibody in which the complementarity determining regions (CDRs) of a human antibody have been replaced by those of a non-human antibody, and where the amino acid sequence of the variable region of the antibody is closest to than for other species.
[0018] The term variant means a sequence of nucleotides, proteins or amino acids different from the sequences specifically identified, in which one or more nucleotides, proteins or amino acid residues are excluded, substituted or added. The variants can be naturally occurring allelic variants, or non-naturally occurring variants. Variants of the identified sequences may retain some or all of the functional characteristics of the identified sequences.
[0019] The term percentage (%) of sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percentage of sequence identity and without considering any conservative substitution as part of the sequence identity. Alignment for purposes of determining the percentage of amino acid sequence identity can be achieved from
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9/44 in various ways, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Preferably, the% of sequence identity values are generated using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program is publicly available from Genentech, Inc. (South San Francisco, CA) or can be compiled from source code, which has been filed with user documentation at the US Copyright Office and is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program must be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are defined by the ALIGN-2 program and do not vary.
[0020] In situations where ALIGN-2 is used for comparisons of amino acid sequences, the% sequence identity of a given A amino acid sequence for, with or against a B amino acid sequence (which can be expressed as a given sequence of amino acids A that has or comprises a certain% amino acid sequence identity for, with or against a given sequence of amino acids B) is calculated as follows: 100 times the X / Y fraction where X is the number of amino acid residues classified as identical matches by the ALIGN-2 sequence alignment program where the alignment of A and B of that program, and where Y is the total number of amino acid residues in B. When the length of amino acid sequence A is not equal to the length of the B amino acid sequence, the% amino acid sequence identity from A to B will not be equal to the% amino acid sequence identity from B to A. Unless otherwise specified the form, all% amino acid sequence identity values used here
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10/44 are obtained using the computer program ALIGN-2.
BRIEF DESCRIPTION OF THE FIGURES
[0021] Figure 1 illustrates the antibody binding of a commercially available murine antiAGE antibody.
[0022] Figure 2 illustrates a chromatogram of a transfected murine anti-AGE monoclonal antibody.
[0023] Figure 3 illustrates a gel electropherogram of a transfected murine anti-AGE monoclonal antibody.
[0024] Figure 4 illustrates the binding of a transfected murine anti-AGE monoclonal antibody to CML-OVA in an enzyme-linked immunosorbent assay.
DETAILED DESCRIPTION
The present invention relates to a humanized monoclonal antibody that binds to an AGE-modified protein or peptide in a cell. Specifically, the anti-AGE antibody binds to a protein or peptide modified with carboxymethyl lysine in a cell. The antibody is suitable for in vivo administration to a human subject and is preferably substantially non-immunogenic to humans. The antibody can optionally be conjugated to a toxin or other agent to induce cell death. The antibody can also be included in a composition with a pharmaceutically acceptable carrier. The antibody is believed to have superior antigen-binding properties compared to comparable commercially available non-human anti-AGE antibodies.
[0026] The humanized monoclonal antibody of the advanced glycation end product includes a heavy chain with 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 with a protein sequence selected from the group consisting of
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11/44 in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. The humanized heavy chain variable domains can 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. Humanized light chain variable domains can 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.
[0027] The anti-AGE antibody binds to proteins or peptides having a modification in the AGE-linked carboxymethyl lysine. Carboxymethyl lysine (also known as N (epsilon) - (carboxymethyl) lysine, N (6) -carboxymethyl lysine, 2-amino-6- (carboxymethylamino) hexanoic acid and CML) is found in proteins or peptides and lipids as a result oxidative stress and chemical glycation. Proteins or peptides modified with carboxymethyl lysine are recognized by the RAGE receptor, which is expressed in a variety of cells. Carboxymethyl lysine has been well studied and products related to carboxymethyl lysine are commercially available. For example, Cell Biolabs, Inc. sells CML-BSA antigens, CML polyclonal antibodies, CML immunoblot kits and competitive CML ELISA kits (www.cellbiolabs.com/cml-assays). CML-ovalbumin (CML-OVA) is a preferred control for checking the binding to the antibody.
[0028] The anti-AGE antibody has a low rate of dissociation from the antibody-antigen complex, or kd (also known as kback or dissociation), preferably at most 6 x 10 ' ³ , 5 x 10' ³ , 1 x 10 ³ , 8 x 10 ⁴ , 5 x 10 ⁴ , 1 x 10 ⁴ , 8 x 10 ⁵ , 5 x 10 ⁵ or 1 x 10 ⁵ (s ¹ ). Preferably, the binding properties of the anti-AGE antibody are superior to those of murine monoclonal carboxymethyl lysine antibody (Clone 318003) available from R&D Systems, Inc. (Minneapolis, MN; Catalog No. MAB3247), illustrated in Figure 1.
[0029] The binding of humanized antibodies can be assessed by
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12/44 example, by dose-dependent binding ELISA or cell-based binding assay. Preferably, the binding of the humanized anti-AGE antibodies is equivalent to or greater than the binding of non-human antiAGE antibodies.
[0030] The anti-AGE antibody can destroy AGE-modified cells through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is a cell-mediated immune defense mechanism, in which an effector cell of the immune system actively smooths a target cell whose antigens on the membrane surface have been linked by specific antibodies. ADCC can be mediated by natural killer cells (NK), macrophages, neutrophils or eosinophils. Effector cells bind to the Fc portion of the bound antibody. Administration of NK cells, such as NK92 cells (a cell line available from NantKwest, Culver City, CA), together with or after administration of anti-AGE antibodies, can improve compilation activity and therefore the effectiveness of anti-AGE antibodies to kill cells. The anti-AGE antibody can also destroy cells modified with AGE through complement-dependent cytotoxicity (CDC). In CDC, the complement cascade of the immune system is triggered by a binding of the antibody to a target antigen.
[0031] The anti-AGE antibody can optionally be conjugated to an agent that causes the destruction of AGE-modified cells. Examples of such agents include toxins, cytotoxic agents, magnetic nanoparticles and magnetic rotating vortex disks.
[0032] A toxin, such as a pore-forming toxin (PFT) (Aroian R. et al., Pore-Forming Toxins and Cellular Non-Immune Defenses (CNIDs), Current Opinion in Microbiology, 10: 57 to 61 (2007 )), conjugated to an anti-AGE antibody, can be injected into a patient to selectively target and remove modified cells
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13/44 with AGE. The anti-AGE antibody recognizes and binds to cells modified with AGE. Then, the toxin causes formation of pores on the cell surface and subsequent cell removal through osmotic lysis.
[0033] Magnetic nanoparticles conjugated to the antiAGE antibody can be injected into a patient to target and remove AGE modified cells. Magnetic nanoparticles can be heated by applying a magnetic field in order to selectively remove AGE modified cells.
[0034] As an alternative, rotating vortex magnetic disks, which are magnetized only when a magnetic field is applied to prevent self-aggregation that can block blood vessels, begin to rotate when a magnetic field is applied, causing rupture of the target cell membrane. Magnetic rotating vortex discs, conjugated with anti-AGE antibodies, specifically target cell types modified with AGE, without removing other cells.
[0035] A humanized anti-AGE monoclonal antibody or a variant thereof may include a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, including post-translational modifications. A heavy chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity can contain substitutions (for example, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence maintains the ability to bind to AGE. Substitutions, insertions or deletions can occur anywhere in the sequence.
[0036] A humanized anti-AGE monoclonal antibody or a
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14/44 variant thereof may include a variable region of the heavy chain that has at least 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, including its post-translational modifications. A variable region with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity may contain substitutions (for example, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence maintains the ability to bind to AGE. Substitutions, insertions or deletions can occur anywhere in the sequence.
[0037] A humanized anti-AGE monoclonal antibody or a variant thereof may include a light chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, including their post-translational modifications. A light chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity can contain substitutions (for example, conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-AGE antibody including that sequence maintains the ability to bind to AGE. Substitutions, insertions or deletions can occur anywhere in the sequence.
[0038] A humanized anti-AGE monoclonal antibody or a variant thereof may include a variable region of the light chain with at least 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20, including post-modifications translational. A variable region with
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15/44 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity may contain substitutions (for example, conservative substitutions), insertions , or deletions related to the reference sequence, but an anti-AGE antibody including that sequence maintains the ability to bind to AGE. Substitutions, insertions or deletions can occur anywhere in the sequence.
[0039] Antibody fragments can be used in place of whole antibodies. Preferably, the fragments are derived from an antibody composed of 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. Antibodies can be divided into smaller fragments digestion with enzymes. Papain digestion cleaves the N-terminal side of the inter-heavy chain disulfide bridges to produce Fab fragments. Fab fragments include the light chain and one of the two N-terminal domains of the heavy chain (also known as the Fd fragment). Pepsin digestion cleaves the C-terminal side of the heavy inter-chain disulfide bridges to produce F (ab ') 2 fragments. F (ab ') 2 fragments include both light chains and two disulfide-linked N-terminal domains. Digestion with pepsin can also form the Fv (fragment variable) and Fc (crystallizable fragment) fragments. The Fv fragment contains the two N-terminal variable domains. The Fc fragment contains the domains that interact with immunoglobulin receptors in cells and with the initial elements of the complement cascade. Pepsin can also cleave immunoglobulin G before the third heavy chain constant domain (CH3) to produce a large F (abc) fragment and a small
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16/44 pFc 'fragment. Antibody fragments can alternatively be produced recombinantly.
[0040] Humanized antibody sequences can be compared with known antibody sequences to predict their effectiveness. For example, humanized antibody sequences can be analyzed by eye and / or computer modeling to identify sequences that are likely to retain binding to the antigen. Humanized antibody sequences can also be screened for the presence of sequences that increase the possibility of an immunogenic response. For example, the presentation of peptide sequences in the groove of MHC Class II molecules leads to the activation of CD8 + T cells and an immunogenic response. In order to reduce this response, antibodies can be designed to prevent the incorporation of T cell epitopes that can activate T cells, reducing the binding affinity to MHC Class II molecules. Residues within human structures or CDRs can be mutated to the equivalent of the human germline (a process known as the genetic line) to remove potential MHC-II epitopes.
[0041] The anti-AGE antibody can be obtained by humanizing a murine anti-AGE monoclonal antibody. A murine antiAGE monoclonal antibody has the heavy chain protein sequence shown in SEQ ID NO: 1 (the variable domain protein sequence 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 can be produced recombinantly in Chinese Hamster Ovary (CHO) cells. Humanized monoclonal antibodies can be purified after synthesis. For example, antibodies can be purified using MabSelect SuRe Protein A (GE Healthcare).
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[0042] Humanized anti-AGE monoclonal antibodies can be included in a composition with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier includes any and all solvents, dispersion medium, coatings, antibacterial and antifungal agents, isotonic agents and absorption retardants, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or diluents include water, saline, Ringer's solutions and dextrose solution. Supplementary active compounds can also be incorporated into the compositions. Solutions and suspensions used for parenteral administration may include a sterile diluent, such as water for injection, saline, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be placed in ampoules, disposable syringes or vials for multiple doses of glass or plastic.
[0043] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersions. Various excipients can be included in pharmaceutical compositions of antibodies suitable for injection. 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 must be fluid in order to be administered using a syringe. Such compositions must be stable during
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18/44 manufacture and storage and to be preserved against contamination from micro-organisms, such as bacteria and fungi. Various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal, can contain contamination by microorganisms. 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 agents such as aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating antibodies and, optionally, other therapeutic components, in the required amount in an appropriate solvent with one or a combination of ingredients as needed, followed by sterilization. Methods of preparing sterile solids for the preparation of sterile injectable solutions include vacuum drying and lyophilization to produce a solid.
[0044] For administration by inhalation, antibodies can be delivered as an aerosol spray from a nebulizer or a pressurized container containing a suitable propellant, for example, a gas such as carbon dioxide. Antibodies can also be delivered by inhalation as a dry powder, for example, using the iSPERSE ™ inhaled drug delivery platform (PULMATRIX, Lexington, MA).
An appropriate dosage level for each type of antibody will generally be about 0.01 to 500 mg per kg of the patient's body weight. Preferably, the dosage level will be from about 0.1 to about 250 mg / kg; more preferably about 0.5 to about 100 mg / kg. A suitable dosage level can 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 dosage can be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg / kg. The antibodies can be administered in a
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19/44 regime 1 to 4 times a day, such as once or twice a day. Antibodies generally have a long half-life in vivo, which can reduce the administration regimen to once a day, once a week, once every two or three weeks, once a month or once every 60 days. to 90 days.
[0046] An individual who receives an anti-AGE antibody can be tested to determine whether the antibody has effectively removed AGE-modified cells. The presence of AGE modified cells can be determined by measuring markers that are associated with AGE modification, such as p16 ^INK4a . Antibody administration and subsequent tests can be repeated until the desired therapeutic result is achieved.
[0047] Unit dosage forms can be created to facilitate administration and uniformity of dosage. The unit dosage form refers to physically discrete units suitable as single dosages for the individual to be treated, containing a therapeutically effective amount of one or more types of antibodies in association with the required pharmaceutical carrier. Preferably, the unit dosage form is in a sealed container and is sterile.
[0048] Any human individual who has been diagnosed with a pathological condition, disease or disorder associated with AGEs or AGE modified cells can be treated by the methods described herein. Examples of pathological conditions, diseases or disorders that can be treated with humanized anti-AGE monoclonal antibodies include Alzheimer's disease, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), chronic obstructive pulmonary disease (COPD), Huntington's chorea, fibrosis idiopathic pulmonary, muscular dystrophy (including Becker, Duchenne, Limb-Girdle and Yamamoto muscular dystrophy), macular degeneration, cataracts, diabetic retinopathy,
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20/44 Parkinson's disease, progeria (including Werner's syndrome and Hutchinson Gilford's 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, cervix cancer, bladder cancer, rectal cancer, esophageal cancer, cancer liver, mouth and throat cancer, multiple myeloma, ovarian cancer, stomach cancer, pancreatic cancer, and retinal blastoma), cancer therapy-related deficiency or side effects of cancer therapy, hypertension, glaucoma, osteoporosis, sarcopenia , cachexia, stroke, myocardial infarction, atrial fibrillation, transplant rejection, diabetes mellitus - Type I, diabetes mellitus - Type II, radiation exposure, side effects of treatment for HIV, exposure to chemical weapons, poisoning, inflammation, nephropathy, Lewy body dementia, disease caused by prions (including bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, scrapie, chronic weight loss disease, kuru and fatal familial insomnia), lordoififosis, autoimmune disorders, loss of adipose tissue, psoriasis, Crohn's disease, asthma, the physiological effects of aging (including cosmetic effects, such as wrinkles, age spots, hair loss, reduction of subcutaneous fat tissue and thinning of the skin), idiopathic myopathy (including, for example, idiopathic inflammatory myopathy, idiopathic inflammatory myositis, polymyositis, dermatomyositis, sporadic inclusion body myositis and juvenile myositis), multiple sclerosis, optic neuromyelitis (NMO, Devic's disease or Devic), epilepsy and adrenoleukodystrophy (ALD, X-linked adrenoleukodystrophy, X-ALD, cerebral ALD or cALD).
[0049] A particularly preferential treatment group includes
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21/44 individuals who have been diagnosed with a pathological condition, disease or disorder associated with AGEs or AGE-modified cells, but who are unable to receive conventional treatments. For example, metastatic cancer has been recognized as a condition associated with AGE-modified cells. A patient with metastatic cancer may not be able to undergo cancer treatments, such as surgery, radiation or chemotherapy due to other diagnoses, physical conditions or complications. For example, pregnant women cannot receive radiation therapy because of the risk of harm to the fetus. Elderly or weakened patients, such as those experiencing cancer cachexia, may not be good candidates for surgery due to the risk of not surviving an invasive procedure. Patients who already have a compromised immune system or a chronic infection may not be able to receive chemotherapy, as many chemotherapy drugs damage the immune system.
[0050] Anti-AGE antibodies can be used in cell separation processes, such as magnetic cell separation. In the magnetic separation of cells, anti-AGE antibodies are attached to the magnetic spheres through a process called coating. The coated magnetic spheres can then specifically bind to AGE modified cells. AGE-modified cells that have bound to anti-AGE antibodies coated in magnetic beads will then respond to an applied magnetic field, allowing AGE-modified cells to be separated from non-AGE-modified cells. Magnetic cell separation can be used to isolate AGE modified cells from tissue samples and fluid samples. Magnetic spheres can be microspheres (0.5 - 500 pm) or nanoparticles (5 - 500 nm). Anti-AGE antibodies coated on magnetic beads can also be used in isolation processes such as immunoassays and
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22/44 immunoprecipitation. Likewise, anti-AGE antibodies coated on magnetic beads can be used to specifically target and separate AGE-modified proteins or peptides from tissue samples and fluid samples.
[0051] Anti-AGE antibodies can be used in cell purification processes, such as immunopanning and immunoadsorption. Purification processes are useful in isolating desirable or unwanted cells from tissue cultures, cell cultures or blood. Cell purification can be used in transplants, such as a bone marrow transplant, or transfusions, such as a blood transfusion. Cell purification is especially useful in autologous stem cell transplantation during chemotherapy to remove metastatic malignant cells and to concentrate beneficial stem cells. Immunopanning or immunoadsorption using an anti-AGE antibody can isolate AGE-modified cells from a tissue culture, cell culture or blood sample.
[0052] The amino acid sequence of a letter corresponding àSEQ ID NO: 1 is MGWTLVFLFLLSVTAGVHSQVQLLQPGAELVKPGASVKLACKASGYL FTTYWMHWLKQRPGQGLEWIGEISPTNGRAYYNARFKSEATLTVDK SSNTAYMQLSSLTSEASAVYYCARSFGNYEFAYWGQGTLVTVSVAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K.
[0053] The amino acid sequence of a letter that corresponds
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23/44 àSEQ ID NO: 2 is MGWTLVFLFLLSVTAGVHSEVQLLESGAEAKKPGASVKLSCKASGYL FTTYWMHWVHQAPGQRLEWMGEISPTNGRAYYNARFKSRVTITVDK SASTAYMELSSLRSEDTAVYYCARSFGNYEFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K.
[0054] The amino acid sequence of a letter corresponding àSEQ ID NO: 3 is MGWTLVFLFLLSVTAGVHSQVQLVQSGAEVKKPGASVKVSCKASGY LFTTYWMHWVRQAPGQRLEWIGEISPTNGRAYYNARFKSRVTITRDT SASTAYMELSSLRSEDTAVYYCARSFGNYEFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K.
[0055] The one letter amino acid sequence that corresponds to SEQ ID NO: 4 is MGWTLVFLFLLSVTAGVHSQVQLVQSGAEVKKPGSSVKVSCKASGY LFTTYWMHWVRQAPGQGLEWMGEISPTNGRAYYNARFKSRVTITELGSTSTAY
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STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K.
[0056] The amino acid sequence of a letter corresponding àSEQ ID NO: 5 is MGWTLVFLFLLSVTAGVHSQVQLVQSGAEVKKPGASVKVSCEASGY LFTTYWMHWVRQAPGQGLEWMGEISPTNGRAYYNARFKSRVTITRD TSINTAYMELSRLRSDDTAVYYCARSFGNYEFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K.
[0057] The one-letter amino acid sequence that corresponds to SEQ ID NO: 6 is QVQLLQPGAELVKPGASVKLACKASGYLFTTYWMHWLKQRPGQGL EWIGEISPTNGRAYYNARFKSEATLTVDKSSNTAYMQLSSLTSEASA VYYCARSG VYYCARSG
[0058] The one letter amino acid sequence corresponding to SEQ ID NO: 7 is EVQLLESGAEAKKPGASVKLSCKASGYLFTTYWMHWVHQAPGQRL EWMGEISPTNGRAYYNARFKSRVTITVDKSASTAYMELSSLRSEDTA
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VYYCARSFGNYEFAYWGQGTLVTVSS.
[0059] The one letter amino acid sequence that corresponds to SEQ ID NO: 8 is QVQLVQSGAEVKKPGASVKVSCKASGYLFTTYWMHWVRQAPGQRL EWIGEISPTNGRAYYNARFKSRVTITRDTSASTAYMELSSLRSEDTAVYYAYAR.
[0060] The one letter amino acid sequence that corresponds to SEQ ID NO: 9 is QVQLVQSGAEVKKPGSSVKVSCKASGYLFTTYWMHWVRQAPGQGL EWMGEISPTNGRAYYNARFKSRVTITADKSTSTAYMELSSLRSEDTAGYTYGARFYGAR.
[0061] The one-letter amino acid sequence that corresponds to SEQ ID NO: 10 is QVQLVQSGAEVKKPGASVKVSCEASGYLFTTYWMHWVRQAPGQGL EWMGEISPTNGRAYYNARFKSRVTITRDTSINTAYMELSRLRSDDTAVYGARS.
[0062] The amino acid sequence of a letter corresponding àSEQ ID NO: 11 is MVSSAQFLGLLLLCFQGTRCDVVMTQTPLSLPVSLGDQASISCRSRQ SLVNSNGNTFLQWYLQKPGQSPKLLIYKVSLRFSGVPDRFSGSGSGT DFTLKISRVEAEDLGLYFCSQSTHVPPTFGGGTKLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC.
[0063] The one letter amino acid sequence corresponding to SEQ ID NO: 12 is
MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLPVTLGQPASISCRSRQS LVNSNGNTFLQWLQQRPGQPPRLLIYKVSLRFSGVPDRFSGSGAGT DFTLTISRVEAEDVGIYVSQTQVSQVSQVSQVGQQVQVQQQQQ
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EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC.
[0064] The amino acid sequence of a letter corresponding to SEQ ID NO: 13 is
MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLSVTPGQPASISCRSRQ SLVNSNGNTFLQWYLQKPGQSPQLLIYKVSLRFSGVPDRFSGSGSG TDFTLKISRVEPEDVGVYYCSQSTHVPPTFGGGTKVEVKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.
[0065] The amino acid sequence of a letter corresponding to SEQ ID NO: 14 is
MVSSAQFLGLLLLCFQGTRCDVVMTQSPLSLPVTLGQPASISCRSRQ SLVNSNGNTFLQWFQQRPGQSPRRLIYKVSLRFSGVPDRFSGSGSD TDFTLRISRVEAEDVGLYYCSQSTHVPPTFGQGTKLEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC.
[0066] The one letter amino acid sequence corresponding to SEQ ID NO: 15 is
MVSSAQFLGLLLLCFQGTRCDIVMTQTPLSLSVTPGQPASISCRSRQ SLVNSNGNTFLQWLLQKPGQPPQLLIYKVSLRFSGVPNRFSGSGSGT DFTLKISRVEAEDVGLYYCSQSTHVPPTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC.
[0067] The amino acid sequence of a letter corresponding to SEQ ID NO: 16 is
DVVMTQTPLSLPVSLGDQASISCRSRQSLVNSNGNTFLQWYLQKPG QSPKLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCS
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QSTHVPPTFGGGTKLEIK.
[0068] The amino acid sequence of a letter corresponding to SEQ ID NO: 17 is
DIVMTQTPLSLPVTLGQPASISCRSRQSLVNSNGNTFLQWLQQRPGQ PPRLLIYKVSLRFSGVPDRFSGSGAGTDFTLTISRVEAEDVGIYFCSQ STHVPPTFGQGTKVEIK.
[0069] The one letter amino acid sequence corresponding to SEQ ID NO: 18 is
DIVMTQTPLSLSVTPGQPASISCRSRQSLVNSNGNTFLQWYLQKPGQ SPQLLIYKVSLRFSGVPDRFSGSGSGTDFTLKISRVEPEDVGVYYCS QSTHVPPTFGGGTKVEVK.
[0070] The one letter amino acid sequence that corresponds to SEQ ID NO: 19 is DVVMTQSPLSLPVTLGQPASISCRSRQSLVNSNGNTFLQWFQQRPG QSPRRLIYKVSLRFSGVPDRFSGSGSDTDFTLRISRVEAEDVGY.
[0071] The amino acid sequence of a letter corresponding to SEQ ID NO: 20 is
DIVMTQTPLSLSVTPGQPASISCRSRQSLVNSNGNTFLQWLLQKPGQ PPQLLIYKVSLRFSGVPNRFSGSGSGTDFTLKISRVEAEDVGLYYCSQ STHVPPTFGGGTKVEIK.
EXAMPLES
[0072] Example 1: Affinity and kinetics of a commercially available anti-AGE antibody
[0073] The affinity and kinetics of a commercially available mouse anti-glycation end product antibody have been studied. An anti-AGE antibody produced against carboxymethyl lysine conjugated to keyhole limpet hemocyanin (Clone 318003) (R&D Systems, Inc., Minneapolis, MN; Catalog No. MAB3247) was obtained. The Na, Na-bis (carboxymethyl) -L-lysine trifluoroacetate salt (Sigma-Aldrich, St. Louis, MO) was
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28/44 used as a model substrate for a cell's AGE-modified protein. The non-marking interaction analysis was performed on a BIACORE ™ T200 (GE Healthcare, Pittsburgh, PA), using a S Series CM5 sensor chip (GE Healthcare, Pittsburgh, PA), with Fc1 defined in white, and Fc2 immobilized with the test antibody (molecular weight 150,000 Da). The running buffer was an HBS-EP buffer (10 mM HEPES, 150 mM NaCI, 3 mM EDTA and 0.05% P-20, pH 7.4), at a temperature of 25 ° C. The software was the BIACORE ™ T200 evaluation software, version 2.0. A double reference (Fc2-1 and buffer injection only) was used in the analysis, and the data was fitted to a 1: 1 Langmuir binding model.
Table 1: Experimental configuration of affinity and kinetics analysis
Association and Dissociation Flow path Fc1 and Fc2 Flow rate (μΙ / min.) 30 Membership time (s) 300 Dissociation time (s) 300 Sample concentration (μΜ) 20 - 5 - 1.25 (x2) - 0.3125 - 0.078 - 0
[0074] Figure 1 illustrates a graph of antibody response versus time. The following values were determined from the analysis: k _a (1 / Ms) = 1,857 x 10 ³ ; kd (1 / s) = 6.781 x 10 ³ ; K _D (M) = 3.651 x 10 ⁶ ; Rmax (RU) = 19.52; and Chi ² = 0.114. Since the Chi ² value of the setting is less than 10% of Rmax, the setting is reliable.
[0075] Example 2: Transient expression of murine anti-AGE monoclonal antibody
[0076] A monoclonal anti-murine AGE antibody was transfected into Chinese hamster ovary (CHO) cells to express and purify enough antibody for evaluation by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis ( SPR). The DNA encoding the sequence of
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29/44 amino acids of the antibody was synthesized. The DNA was cloned into the mammalian transient expression plasmid pD2610-v13 (DNA2.0). [0077] CHO cells adapted to the suspension (Thermo Fisher, UK) were cultured at 2.0 to 3.0 x 10 ⁵ cells / ml at 135 rpm, 8% CO2, 37 ^Q C in serum free media ProCHO -4 (Lonza, Belgium) supplemented with 8 mM of L-glutamine (Thermo Fisher, United Kingdom) and 10 mL / L of hypoxanthine / thymidine (Thermo Fisher, United Kingdom) in 500 mL of ventilated Erlenmeyer flasks (Corning, Netherlands) . The maxipreps of the construct were prepared using a PureLink® HiPure plasmid filter maxiprep kit (Thermo Fisher, UK). The vector DNA was quantified using a NanoDrop Lite spectrophotometer.
[0078] 500 ml of cells at a final density of 1.0 x 10 ⁶ cells / ml were transiently transfected with 1.25 pg / ml of vector DNA and cultured in serum free medium ProCHO-5 (Lonza, Belgium) supplemented with 8 mM L-glutamine (Invitrogen, United Kingdom) and 10 mL / L of hypoxanthine / thymidine (Invitrogen, United Kingdom) in 500 mL ventilated Erlenmeyer flasks (Corning, Netherlands). The cultures were incubated for 8 days at 37 ^Q C, 8% CO2 and 135 rpm, and routinely fed with 7.5% (by volume) Power Feed A (Sartorius, Germany) every 2 to 3 days before harvesting by centrifugation at 4000 rpm, 4 ^Q C for 40 minutes. The transfection produced 612 ml of antibody. [0079] The purification of antibodies was carried out using AKTA chromatography ο equipment (GE Healthcare) at room temperature (19 C ^Q). After centrifugation, the filtered supernatant (0.22 pm) of the cell culture supernatant was applied to an AKTA system equipped with a 1 ml HiTrap Protein A column that was equilibrated with wash buffer. After loading, the column was washed with 20 column volumes of wash buffer. Bound antibody was eluted in steps with 10 column volumes of buffer
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30/44 elution. Figure 2 illustrates the antibody chromatogram at 280 nm. All eluted fractions were neutralized with pH 9.0 Tris buffer. The eluted fractions corresponding to the peak elution were selected for dialysis overnight in PBS at 4 ^Q C.
[0080] The purity of the antibody was assessed using polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE). The antibody was found to be> 95% pure. Figure 3 illustrates the gel electropherogram of the antibody. Under reducing conditions, both the heavy chains and the light chains of the antibody were visible and were observed at the expected molecular weights of approximately 50 and 25 kDa, respectively. Under non-reducing conditions, a single main band was observed. The lack of additional large bands indicated an absence of antibody aggregates.
[0081] The concentration of purified antibodies was assessed by spectrophotometry. The antibody was quantified with a NanoDrop Lite spectrophotometer using the extinction coefficient 205,500 M ' ¹ cnrr ¹ (or 1.0 mg / mL = A280 of 1.37 [assuming a MW = 150,000 Da]) as a standard reference for IgG at A280 , according to the manufacturer's instructions. The 600 ml of murine antibody transfected with a concentration of 0.6 mg / ml was purified to 2.3 ml for a total yield of 1.4 mg of antibody.
[0082] The binding of the transfected antibody was assessed by ELISA. 100 ng / well of CML-OVA / Ν ^ε - (carboxymethyl) lysine-OVA (Circulex, Japan, Catalog No. CY-R2053) were immobilized on a 96-well MaxiSorp® plate in coating buffer (NaHCOs at 0, 05 M brought to pH 9.5 by the addition of NasCOs 0.05 M) at 4 ^Q C overnight. The coating buffer was removed and the plate was washed three times with PBS Tween (PBS-T) (0.1% (by volume) Tween 20). 200 pL per well of 3% skimmed milk (weight / volume) in PBS was added to each well and stirred for 2 hours at room temperature.
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31/44 environment. The plate was then washed three times with PBS-T.
[0083] The antibody was diluted from 1,000 ng / ml to 0.488 ng / ml in incubation buffer (PBS, 1% (weight / volume) BSA). 100 pL per well of the diluted antibody was added to the plate in triplicate and stirred for two hours at room temperature.
[0084] The wells were washed three times with PBS-T. After washing, 100 pL per well of goat anti-mouse HRP (specific Fc) (Bio Rad, Cat. No. 0300-0108P) diluted to 1: 5,000 in incubation buffer was added to all wells and the plate was stirred for one hour at room temperature. The wells were washed three times with PBS-T. After washing, 100 ul of TMB substrate was added to each well and incubated at 37 ^Q C for 10 minutes. 50 µl of 1M HCI was added to each well and the plates were read immediately at 450 nm in a Tecan Sunrise plate reader. Figure 4 illustrates the ELISA of antibody binding to CML-OVA. The values shown in the graph are the average of the readings in triplicate. [0085] ELISA results indicate that the transfected antibody recognizes and binds to the CML-OVA protein, a protein known to be modified with AGE. The results confirm that the antibody sequence is correct and the antibody is active. Similar results would be expected for humanized anti-AGE monoclonal antibodies that include the regions determining the complementarity of these murine antibodies.
[0086] Example 3: Production of humanized antibodies
[0087] An anti-murine 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 heavy and light chain variable domains are shown in SEQ ID NO: 6 and SEQ ID NO: 16, respectively.
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[0088] Murine heavy chain CDR residues were identified using the IMGT and Kabat numbering systems. The V region of the human germline gene closest to the variable region of the murine heavy chain was determined. The direct line databases of human IgG sequences were searched for comparison with the murine heavy chain variable domain using BLAST search algorithms, and the candidate human variable domains were selected from the top 200 BLAST results. These were reduced to four candidates based on a combination of structure homology, maintaining the main structure residues and the canonical loop structure.
[0089] CDRs from the murine heavy chain variable domain were grafted into the four acceptor structures to produce four variants of the humanized heavy chain variable domain. The amino acid sequences of the four variable domains of the humanized heavy chain are shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. The homology of the variable domains of the humanized heavy chain was compared to the murine heavy chain variable domain. The results of the homology comparison are shown in Table 2 below:
Table 2: Heavy chain variable domain homology
Heavy chain variable domain Amino Acids Amino Acids humanized identical consensus SEQ ID NO: 7 82.2% 87.3% SEQ ID NO: 8 81.4% 89.0% SEQ ID NO: 9 81.4% 90.7% SEQ ID NO: 10 79.7% 88.1%
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[0090] In order of homology, SEQ ID NO: 7 is the most similar to the murine heavy chain variable domain, followed by SEQ ID NO: 9, SEQ ID NO: 8 and SEQ ID NO: 10.
[0091] Murine light chain CDR residues were identified using the IMGT and Kabat numbering systems. The V region of the human germline gene closest to the variable region of the murine light chain was determined. The direct line databases of human IgK sequences were searched for comparison with the murine light chain variable domain using BLAST search algorithms, and the candidate human variable domains were selected from the top 200 BLAST results. These were reduced to four candidates based on a combination of structure homology, maintaining the main residues of the structure and the canonical loop structure.
[0092] The murine light chain variable domain CDRs were grafted into the four acceptor structures to produce four humanized light chain variable domain variants. The amino acid sequences of the four variable domains of the humanized light chain are shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. The homology of the humanized light chain variable domains was compared with the murine heavy chain variable domain. The results of the homology comparison are shown in Table 3 below:
Table 3: Homology of the light chain variable domain
Light chain variable domain Amino Acids Amino Acids humanized identical consensus SEQ ID NO: 17 86.6% 93.8% SEQ ID NO: 18 88.4% 94.6%
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Light chain variable domain Amino Acids Amino Acids humanized identical consensus SEQ ID NO: 19 87.5% 94.6% SEQ ID NO: 20 88.4% 92.9%
[0093] In order of homology, SEQ ID NO: 18 is the most similar to the murine light chain variable domain, followed by SEQ ID NO: 20, SEQ ID NO: 19 and SEQ ID NO: 17.
[0094] Humanized variants of the heavy and light chain variable domain have been checked to determine whether they have been humanized according to the World Health Organization (WHO) definition of a humanized antibody. WHO considers that an antibody should be humanized if the amino acid sequence of the variable region is closer to humans than to other species. SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 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 to 307). All humanized variable domains were more human than murine. Therefore, all humanized variable domains meet the WHO definition of humanized antibodies.
[0095] The heavy and light chain variable domains of the murine antibody and the eight variant sequences of the humanized heavy and light chains were screened for MHC II-binding peptides to determine whether the humanization process removed highly affinity peptide sequences using in silico algorithms. The human heavy chain germline sequences
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IGHV1-46 and IGHV1-3 and the human light chain germline sequences IGKV2-30 and IGKV2-29 were also analyzed for comparison. The sequences were screened for the following 8 alleles, which represent more than 99% of the world population and are the set of standard alleles used for the prediction of MHC Class II epitopes: DRB1 * 01: 01; DRB1 * 03: 01; DRB1 * 04: 01; DRB1 * 07: 01; DRB1 * 08: 02; DRB1 * 11: 01; DRB1 * 13: 02; DRB1 * 15: 01.
The murine heavy chain variable domain had two high-affinity T cell epitope nuclei (IC 50 <50 nM). The sequence of the human germline of IGHV1 -46 and SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 10 each had a potential T cell epitope. The sequence of the human germline of IGHV1-3 and SEQ ID NO: 8 each had two potential T cell epitopes. Since human germline sequences are unlikely to be immunogenic, the potential T cell epitopes may be an excessive prediction of the MHC Class epitope software. II. Potential T cell epitopes are most likely epitopes of regulatory T cells, 0 which would be beneficial for sequences.
The murine light chain variable domain and SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 each had two high-affinity T cell epitope nuclei (IC50 <50 nM) and a potential T cell epitope. The IGKV2-30 human germline sequence did not have potential T cell epitopes. The IGKV2-29 human germline sequence had two potential T cell epitopes. as with variable heavy chain sequences, potential T cell epitopes may be an over-prediction of MHC Class II epitope software, but are likely to be beneficial regulatory T cell epitopes.
[0098] Post-translational modifications of murine and humanized antibodies have been studied. The N-linked glycosylation motif
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NXS I T, where X is any amino acid except 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 be prone to deamidation of asparagines in aspartic acid. The SNG motif was present in CDR1 of all light chains. Although this reason is potentially immunogenic, no substitution was made, as it occurred only in the CDR.
[0099] Murine heavy chain and light chain signal peptides have been identified. These signal peptides can result in higher levels of expression in Chinese hamster ovary (CHO) cells. The heavy chain signal peptide is included in the murine heavy chain (SEQ ID NO: 1) and the four humanized heavy chains (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 ). The light chain signal peptide is included in the murine light chain (SEQ ID NO: 11) and in the four humanized light chains (SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 ).
[0100] The structures of the variable domain binding sites were modeled using DNASTAR NovaFold, a protein structure prediction software based on l-Tasser. NovaFold uses l-Tasser algorithms that combine threading and trend-free folding technologies to build complete and accurate 3D atomic models of proteins with previously unknown structures. Analysis of protein structures indicated that combinations of heavy and light chain variable domains SEQ ID NO: 7-SEQ ID NO: 17, SEQ ID NO: 7-SEQ ID NO: 18, SEQ ID NO: 8- SEQ ID NO: 20 and SEQ ID NO: 9-SEQ ID NO: 18 appear to have the structure closest to the combination of murine heavy and light chain variable domains SEQ ID NO: 5-SEQ ID NO: 16. In general , humanized variants that contain the variable domain of
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37/44 light chain with the sequence shown in SEQ ID NO: 18 had better structures than those containing other light chain variable domains. Likewise, humanized variants containing the heavy chain variable domain having the sequence shown in SEQ ID NO: 7 had better structures than those containing other heavy chain variable domains.
[0101] Example 4 (prophetic): Future antibody studies
[0102] Each of the heavy chain variable domains (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10) is synthesized in the structure with a constant domain sequence of the human lgG1 isotype . The entire heavy chain sequence is codon-optimized (DNA2.0, USA) and the DNA sequence is verified. The amino acid sequence of the lgG1 constant domain (G1m17.1 allotype) is shown below:
[0103] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK.
[0104] Each of the light chain variable domains (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20) is synthesized in frame with an IgK isotype constant domain sequence human. The entire light chain sequence is codon-optimized (DNA2.0, USA) and the DNA sequence is verified. The amino acid sequence of the IgK constant domain (Km3 allotype) is shown below:
[0105] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
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38/44
VTHQGLSSPVTKSFNRGEC.
[0106] Each of the variant strands is verified by DNA sequencing analysis. Then, transient transfection and expression of each of the humanized antibodies is performed. A chimeric antibody is expressed for use as a positive control and contains the murine variable domains and the human Ig constant domains. Sixteen humanized variants are expressed that contain the humanized heavy and light chain variable domains and the human Ig constant domains, as shown in Table 4 below:
Table 4: Combinations of chimeric and humanized antibody variants
Chimeric antibody SEQ ID NO: 6-SEQ ID NO: 16 Humanized variants SEQ ID NO: 7 -SEQ ID NO: 17 SEQ ID NO: 7 -SEQ ID NO: 18 SEQ ID NO: 7 -SEQ ID NO: 19 SEQ ID NO: 7 -SEQ ID NO: 20 SEQ ID NO: 8 -SEQ ID NO: 17 SEQ ID NO: 8 -SEQ ID NO: 18 SEQ ID NO: 8 -SEQ ID NO: 19 SEQ ID NO: 8 -SEQ ID NO: 20 SEQ ID NO: 9 -SEQ ID NO: 17 SEQ ID NO: 9 -SEQ ID NO: 18 SEQ ID NO: 9 -SEQ ID NO: 19 SEQ ID NO: 9 -SEQ ID NO: 20 SEQIDNQ: 10-SEQ ID NO: 17 SEQIDNO: 10-SEQ ID NO: 18 SEQIDNO: 10-SEQ ID NO: 19 SEQIDNO: 10-SEQ ID NO: 20
[0107] Example 5 (prophetic): Treatment of sarcopenia
[0108] An elderly patient is diagnosed with sarcopenia. It receives a humanized anti-AGE monoclonal 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. The antibody is administered intravenously at a dose of 5 mg / kg once a week. The antibody is specifically targeted and kills cells that express advanced glycation end products on the cell surface, such as senescent cells. Treatment effectiveness is determined by measuring levels of
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39/44 p16 ^INK4a of the patient before and after administration of the antibody. The patient does not develop an immune response to the antibody. The patient's sarcopenia improves as evidenced by an increase in muscle mass.
[0109] Example 6 (prophetic): Treatment of osteoarthritis
[0110] A patient is diagnosed with osteoarthritis. It is administered a composition comprising a pharmaceutically acceptable carrier and a humanized anti-AGE monoclonal antibody with a heavy chain variable sequence with 98% sequence identity to SEQ ID NO: 7 and a light chain variable region with 98% sequence identity to SEQ ID NO: 18. The antibody is administered orally at a dose of 10 mg / kg once daily. The antibody is specifically targeted and kills cells that express end products of advanced glycation on the cell surface, such as senescent chondrocytes. Treatment effectiveness is determined by measuring the patient's p16 ^INK4a levels before and after administration of the composition. The patient does not develop an immune response to the composition that contains the antibody. The patient's osteoarthritis improves as evidenced by a decrease in joint pain.
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Uptake and Degradation of Glucose-modified Proteins: A Potential Mechanism for the Removal of Senescent Macromolecules, Proc. Natl. Acad. Sci. USA, Vol. 82, 5588, 5591 (1985).
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[0135] 25. What is sarcopenia , available online at www.iofbonehealth.org/what-sarcopenia (2014).
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43/44 therapeutic target for aging and age-related diseases, Clinical Pharmacology & Therapeutics, Vol. 93 (1), p. 105 to 116 (December 5, 2012).
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Endproducts on Erythrocyte Cell Surface Induce Receptor-Mediated Phagocytosis by Macrophages, J. Exp. Med., Vol. 166, 539, 545 (1987). [0148] 38. Fielding, R. A., et al., Sarcopenia: an undiagnosed condition in older adults. Current consensus definition: prevalence, etiology, and consequences, Journal of the American Medical Directors Association, Vol. 12 (4), p. 249 to 256 (May 2011).
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[0153] 43. Hamers-Casterman, C. et al., Naturally occurring
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44/44 antibodies devoid of light chains, Nature, Vol. 363, p. 446 to 448 (June 3, 1993).
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权利要求:
Claims (20)
[1]
1. Humanized monoclonal antibody of the final product of advanced glycation for cell separation processes, characterized by the fact that the antibody comprises 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.
[2]
2. Humanized monoclonal antibody of the final product of advanced glycation for use in the treatment of a pathological condition, disease or disorder associated with AGEs or AGE modified cells, characterized by the fact that the antibody comprises a heavy chain comprising a sequence of amino acids having at the minus 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid 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 comprising an amino acid sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
[3]
3. Use of a humanized monoclonal antibody from the end product of advanced glycation to manufacture a drug for the treatment of a pathological condition, disease or disorder associated with AGEs or AGE-modified cells,
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2/7 characterized by the fact that the antibody comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid 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 comprising an amino acid sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.
[4]
4. Method for treating an individual who has been diagnosed with a pathological condition, disease or disorder associated with AGEs or AGE-modified cells, characterized by the fact that it comprises:
administering to the subject a composition comprising a humanized monoclonal antibody of the final advanced glycation product, wherein the antibody comprises a heavy chain comprising an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid 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 comprising an amino acid sequence with at least 90% sequence identity,
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3/7 preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 , SEQ ID NO: 14 and SEQ ID NO: 15.
[5]
5. Humanized monoclonal antibody of the final product of advanced glycation, characterized by the fact that it comprises:
a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, the light chain comprises an amino acid sequence with at least 90 % sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 and the antibody binds to a protein or peptide modified with carboxymethyl lysine.
[6]
6. Humanized monoclonal antibody of the final product of advanced glycation, characterized by the fact that it comprises:
a heavy chain, having a heavy chain variable region, and a light chain, having a light chain variable region, wherein the heavy chain variable region comprises a
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4/7 amino acid sequence with at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group that consists of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 and the antibody binds to a protein or peptide modified with carboxymethyl lysine.
[7]
7. Humanized monoclonal antibody of the final product of advanced glycation, characterized by the fact that it comprises:
a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence having at least one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, the light chain comprises an amino acid sequence having at least one amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, and the antibody binds to a protein or peptide modified with carboxymethyl lysine.
[8]
8. Humanized monoclonal antibody of the final product of
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5/7 advanced glycation, characterized by the fact that it comprises:
a heavy chain, having a heavy chain variable region, and a light chain, having a light chain variable region, wherein 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 NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, the variable region of the light chain comprises an amino acid sequence having at least one amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, and the antibody binds to a protein or peptide modified with carboxymethyl lysine.
[9]
9. Antibody according to any one of the preceding claims, characterized by the fact that the antibody binds to CML-ovalbumin.
[10]
10. Antibody according to any of the preceding claims, characterized by the fact that the antibody is substantially non-immunogenic to humans.
[11]
11. Antibody according to any one of the preceding claims, characterized by the fact that the antibody has a dissociation rate (kd) of a maximum of 6 x 10 ³ (s ^_1 ).
[12]
12. Antibody according to any one of the preceding claims, characterized by the fact that the antibody is conjugated to an agent that causes the destruction of AGE-modified cells.
[13]
13. Antibody according to any of the preceding claims, characterized by the fact that the agent comprises at least one member selected from the group that
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6/7 consists of toxins, cytotoxic agents, magnetic nanoparticles and magnetic rotating vortex disks.
[14]
14. Antibody according to any of the preceding claims, characterized in that the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with SEQ ID NO: 7 and the light chain variable region comprises an amino acid sequence having at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity, with SEQ ID NO: 18.
[15]
15. Antibody according to any of the preceding claims, characterized by the fact that the heavy chain variable region comprises SEQ ID NO: 7, and the light chain variable region comprises SEQ ID NO: 18.
[16]
16. Composition, characterized by the fact that it comprises:
the humanized monoclonal antibody of the final advanced glycation product as defined in any of the preceding claims, and a pharmaceutically acceptable carrier.
[17]
17. Composition according to any one of the preceding claims, characterized by the fact that the composition is in unit dosage form.
[18]
18. Composition according to any one of the preceding claims, characterized by the fact that the composition is sterile.
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7/7
[19]
19. Antibody, use or method according to any of the preceding claims, characterized by the fact that the pathological condition, disease or disorder associated with AGEs or AGE-modified cells is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, chronic obstructive pulmonary disease, Huntington's chorea, idiopathic pulmonary fibrosis, muscular dystrophy, macular degeneration, cataracts, diabetic retinopathy, Parkinson's disease, progeria, vitiligo, cystic fibrosis, atopic dermatitis, eczema, arthritis, atherosclerosis, cancer, cancer metastatic, therapy-related deficiency or side effects of cancer therapy, hypertension, glaucoma, osteoporosis, sarcopenia, cachexia, stroke, myocardial infarction, atrial fibrillation, transplant rejection, diabetes mellitus - Type I, diabetes mellitus - type II , radiation exposure, side effects of HIV treatment, exposure to chemical weapons, poisoning, inflammation, nephropathy, Lewy body dementia, prion disease, lordoyphiphosis, autoimmune disorders, loss of adipose tissue, psoriasis, Crohn's disease, asthma, physiological effects of aging, idiopathic myopathy, multiple sclerosis, optic neuromyelitis, epilepsy, and adrenoleukodystrophy.
[20]
20. Method according to any one of the preceding claims, characterized by the fact that it additionally comprises:
test the individual for the effectiveness of the administration; and optionally, repeat the administration.

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法律状态:
2021-08-03| B11A| Dismissal acc. art.33 of ipl - examination not requested within 36 months of filing|

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2021-10-19| B11Y| Definitive dismissal - extension of time limit for request of examination expired [chapter 11.1.1 patent gazette]|

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2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762485246P| true| 2017-04-13|2017-04-13|

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US62/485,246|2017-04-13|

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