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EP1086136A1 - Anticorps monoclonaux specifiques de produits finaux de glycosylation avancee derives de groupes guanidino dans des echantillons biologiques - Google Patents

Anticorps monoclonaux specifiques de produits finaux de glycosylation avancee derives de groupes guanidino dans des echantillons biologiques

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Publication number
EP1086136A1
EP1086136A1 EP99928516A EP99928516A EP1086136A1 EP 1086136 A1 EP1086136 A1 EP 1086136A1 EP 99928516 A EP99928516 A EP 99928516A EP 99928516 A EP99928516 A EP 99928516A EP 1086136 A1 EP1086136 A1 EP 1086136A1
Authority
EP
European Patent Office
Prior art keywords
age
antibody
ages
monoclonal antibody
serum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99928516A
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German (de)
English (en)
Inventor
Henry W. Founds
Peter Foiles
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Synvista Therapeutics Inc
Original Assignee
Alteon Inc
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Publication date
Application filed by Alteon Inc filed Critical Alteon Inc
Publication of EP1086136A1 publication Critical patent/EP1086136A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates generally to the detection and measurement of nonenzymatically glycosylated proteins, and particularly to methods and associated materials for the detection and measurement of guanidino group-derived advanced glycosylation endproducts formed by the reaction of 3-deoxyglucosone with the guanidino group of arginine.
  • Glucose and other reducing sugars attach non-enzymatically to the amino groups of proteins and other biomolecules in a concentration-dependent manner. Over time, these initial Amadori adducts can undergo further rearrangements, dehydrations and fragmentations, as well as cross-linking with other protein groups, to accumulate as a family of complex structures referred to as Advanced Glycosylation Endproducts (AGEs).
  • AGEs also known as Maillard or browning products, form in stored food and during the cooking process as a consequence of the reactions of food sugars and proteins with other food components. They are responsible for both the yellow- brown colors of cooked foodstuffs as well as changes in the texture of foods, the cross-linking reactions tending to impart increased toughness.
  • the "family" of AGEs includes species which can be isolated and characterized by chemical structure, some being quite stable, while others are unstable or reactive.
  • AGEs Several structurally identified AGEs have been previously described which arise from the interaction of various biologically-relevant sugars, including glucose, ribose and fructose, with proteins, lipids and nucleic acids. Examples include 2-(2- furoyl)-4(5)-(2-furanyl)-lH-imidazole [See U.S. Patent No. 4,665, 192], pentosidine, AFGP, pyrraline, cypentodine, etc. Numerous AGE structures have not been characterized nor structurally identified.
  • a monoclonal antibody to compound S17 was subsequently produced and used to quantitate and immunohistochemically locate imidazolones in diabetic tissues from rodents and humans (Niwa et al. , 1997, FEBS Letters 407:297-302; Niwa et al. , 1997, J. Clin. Invest. 99: 1272-1280).
  • Al-Abed et al. (Model Studies of the Maillard Reaction of Arg-Lys with D-Ribose, Bioorganic & Medicinal Chemistry Letters 5:2929-2930) described an AGE formed from adjacent arginine and lysine residues, in which the lysine has reacted with a reducing sugar to form the Amadori product.
  • the Amadori product dehydrates to form an Amadori product dione intermediate, which then forms an adduct with the guanidino group of the adjacent arginine, subsequently being hydrated to form a cyclic 2-amino-4,5-dihydroimidazole adduct structure (referred to as ALI, for arg-lys imidazole).
  • ALI cyclic 2-amino-4,5-dihydroimidazole adduct structure
  • AGE advanced glycosylation endproduct
  • the antibodies or fragments thereof do not react with amino-5-(2,3,4-trihydroxybutyl)- 4-imidazolone, pentosidine, carboxymethyllysine, ribose-derived advanced glycosylation endproducts, nor with the reaction product of 3-deoxyglucosone with L-lysine or aminoguanidine, nor of L-arginine with methylglyoxal.
  • a preferred embodiment is a monoclonal antibody or antigen-binding fragment thereof which demonstrates the immunological binding characteristics as described above, such as monoclonal antibody 4E1 as produced by hybridoma F52-4E1, deposited with the American Type Culture Collection (ATCC) on March 31, 1998, and assigned Accession Number HB- 12500.
  • monoclonal antibody 4E1 as produced by hybridoma F52-4E1, deposited with the American Type Culture Collection (ATCC) on March 31, 1998, and assigned Accession Number HB- 12500.
  • said monoclonal antibody or antigen-binding fragment thereof can have an immunological binding characteristic, which characteristic is selected from the group consisting of reactivity with serum-AGE proteins, hemoglobin- AGE, serum lipid-AGEs, serum-AGE peptides, LDL-AGE, and collagen- AGE.
  • the monoclonal antibody is humanized or a chimeric human- murine antibody.
  • Therapeutic compositions including the antibody or active fragments thereof, or agonists and cognate molecules, or alternately, antagonists of the same, and methods of use of such compositions in the prevention, diagnosis or treatment of disease using these compositions are also included, wherein an effective amount of the composition is administered to a patient in need of such treatment.
  • the antigen-binding fragment of the monoclonal antibody can be a single chain Fv fragment, an F(ab') fragment, an F(ab) fragment, and an F(ab') 2 fragment, or any other antigen-binding fragment.
  • the monoclonal antibody or fragment thereof is a murine IgG isotype antibody.
  • the invention extends to the hybridoma that produces monoclonal antibody 4E1, which hybridoma is deposited with the ATCC as indicated hereinbefore.
  • the monoclonal antibody of the invention advantageously binds to in vzr ⁇ -produced AGEs derived from reactions with 3DG or glucose. Accordingly, in another aspect, the invention is directed to a method for detecting the presence of advanced glycosylation endproducts (AGEs) in a biological sample.
  • the method comprises contacting a sample suspected of containing guanidino group-derived AGEs with the monoclonal antibody of the invention or antigen binding fragment thereof under conditions which allow for the formation of reaction complexes comprising the monoclonal antibody or antigen binding fragment thereof and the AGEs; and detecting the formation of such reaction complexes comprising the monoclonal antibody or antigen binding fragment thereof and AGEs in the sample. Detection of the formation of reaction complexes indicates the presence of AGEs in the sample.
  • sample molecules may be allowed to bind or adhere to a solid support and any AGE-modified molecules so immobilized may be recognized by formation of reaction complexes with the monoclonal antibody of the present invention or an antigen-binding fragment thereof, through subsequent assay steps to detect reaction complexes.
  • the monoclonal antibody or antigen-binding fragment thereof is bound to a solid phase support, for instance as the first component of a "sandwich-type" assay for AGE-modified molecules reactive with the immobilized monoclonal antibody of the present invention, or an antigen-binding fragment thereof, wherein the second immunological binding partner may be a polyclonal or a monoclonal antibody, or a mixture thereof, including without limitation the monoclonal antibody of the present invention.
  • the sample is contacted with a labelled advanced glycosylation endproduct (AGE), and unbound substances are removed prior to detecting the formation of reaction complexes in a competitive assay format.
  • AGE advanced glycosylation endproduct
  • reaction complexes with the sample is detected by observing a decrease in the amount of labelled AGE bound in the assay.
  • the formation of reaction complexes can be observed by detecting the binding of a labelled anti-AGE antibody or an antibody to an AGE carrier, such as but not limited to albumin, hemoglobin, low density lipoprotein, and the like, to the complex of the monoclonal antibody or antigen-binding fragment thereof and the AGE.
  • an AGE is bound to a solid phase support.
  • the sample is contacted with said immobilized AGE bound to the solid phase support, in the presence of the monoclonal antibody of the present invention or an antigen-binding fragment thereof.
  • the monoclonal antibody or antigen-binding fragment thereof is labelled either directly or by further assay steps using available reagents that specifically recognize the monoclonal antibody of the present invention or an antigen-binding fragment thereof. Formation of reaction complexes with AGE-modified molecules in the sample is detected by observing a decrease in the amount of label complexed to the solid phase support.
  • the methods for detecting the presence of guanidino group-derived AGEs in a sample according to the invention are useful for evaluating the level of AGEs in a biological sample. Accordingly, the invention is further directed to a method for evaluating the level of AGEs in a biological sample, which comprises detecting the formation of reaction complexes in a biological sample; and evaluating the amount of reaction complexes formed, which amount of reaction complexes corresponds to the level of AGEs in the biological sample.
  • the level of guanidino group-derived AGEs in a sample can have important diagnostic or prognostic value. Accordingly, the invention is further directed to a method for detecting or diagnosing the presence of a disease associated with elevated AGE levels in a mammalian subject comprising evaluating the level of AGEs in a biological sample from a mammalian subject; and comparing the level detected to a level of AGEs normally present in the mammalian subject. An increase in the level of guanidino group-derived AGEs as compared to normal levels indicates a disease associated with elevated levels of AGEs.
  • the invention relates to a method for monitoring the course of a disease associated with elevated AGE levels in a mammalian subject comprising evaluating the level of AGEs in a series of biological samples obtained at different times from a mammalian subject.
  • An increase in the level of AGEs over time indicates progression of the disease, and a decrease in the level of AGEs over time indicates regression of the disease.
  • the invention in another embodiment, relates to a method for monitoring a therapeutic treatment of a disease associated with elevated AGE levels in a mammalian subject comprising evaluating the levels of AGEs in a series of biological samples obtained at different times from a mammalian subject undergoing a therapeutic treatment for a disease associated with elevated AGE levels. A decrease in the level of AGEs over time indicates an effective therapeutic outcome.
  • the invention advantageously provides convenient test kit formats for practicing the foregoing methods. Accordingly, the invention provides a test kit for measuring the presence or amount of in vzr ⁇ -derived guanidino-group-derived AGEs in an analyte.
  • Such a kit can comprise a monoclonal antibody of the invention or an antigen- binding fragment thereof of the invention; means for detecting the formation of reaction complexes between the monoclonal antibody or antigen-binding fragment thereof and AGEs; other reagents; and directions for use of the kit.
  • the test kit can further comprise preparation of an AGE or AGEs, or molecules modified by an AGE or AGEs, recognized by the monoclonal antibody, e.g. , wherein said AGE molecules are irreversibly associated with a solid phase.
  • test kit can further comprise a labelled anti-AGE antibody or antigen-binding fragment thereof, which labelled anti-AGE antibody is reactive with in v vo-produced AGEs, or directly reactive with the analyte molecule whose degree of AGE-modif ication is to be determined, including for instance a labelled anti-low density lipoprotein antibody.
  • the antibodies of the present invention may be used therapeutically in the treatment of a disease or disorder characterized by excessive accumulation of guanidino group-derived AGEs, by exposing a patient's serum to the antibodies in order to sequester AGEs from circulation, either in vivo or ex vivo.
  • a primary object of the invention is to provide a monoclonal antibody reactive with in vz ' vo-produced AGEs derived from structures containing a guanidino group.
  • a further object of the invention is to provide an indefinite source of an antibody reactive with in vz ' v ⁇ -produced, guanidino group-derived AGEs, which antibody has particular immunological binding characteristics that render it particularly useful for this purpose.
  • a still further object of the invention is to provide therapeutic compositions and corresponding methods for treating conditions characterized by abnormal levels of AGEs which are based on or include the antibodies of the present invention.
  • FIGURE 1 depicts the specificity of the 4E1 antibody to products of the reaction between 3-deoxyglucosone and various guanidino group-containing compounds, and controls.
  • FIGURE 2 depicts the reverse phase HPLC chromatographic separation and competitive ELISA data of the products of the reaction of 3-deoxyglucosone with N e -benzoyl-L-arginine amide, incubated at 37°C for 48 hours in 200 mM sodium phosphate buffer, pH 7.4. Seven fractions, as indicated in Figure 2A, were collected. The ELISA data on the seven fractions is shown in Figure 2B
  • FIGURE 3 depicts the reverse phase HPLC chromatographic separation and competitive ELISA data of the products of the reaction of 3-deoxyglucosone with N € -benzoyl-L-arginine amide, incubated at 50 °C for 4 weeks in 200 mM sodium phosphate buffer, pH 7.4. Eleven fractions, as indicated in Figure 3A, were collected. The ELISA data on the eleven fractions is shown in Figure 3B.
  • the present invention is directed to antibodies, or antigen-binding fragments thereof, which are reactive with in vz ' v ⁇ -produced, guanidino group-derived advanced glycosylation endproducts (AGEs) which arise from the reaction of 3- deoxyglucosone (3DG) with arginine and other guanidino group-containing molecules.
  • AGEs guanidino group-derived advanced glycosylation endproducts
  • the antibodies also react with AGEs formed from glucose.
  • the antibody or antigen-binding fragment thereof of the present invention demonstrate the immunological binding characteristics of monoclonal antibody 4E1 as produced by hybridoma F52-4E1, deposited with the American Type Culture Collection (ATCC) on March 31, 1998, and assigned Accession Number HB- 12500.
  • ATCC American Type Culture Collection
  • the invention advantageously provides an indefinitely prolonged cell source of a monoclonal antibody of the invention: the hybridoma.
  • the invention further relates to diagnostic assay methods and kits that comprise the monoclonal antibody of the invention and to therapeutic methods based thereon.
  • immunological binding characteristics refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.
  • R When R is hydrogen, the compound is guanidine; when methyl, the compound is methylguanidine, etc.
  • Methylguanidine is a circulating metabolite present at elevated levels in individuals with reduced renal function.
  • guanidino group-derived refers to compounds which form from the reaction with guanidino group-containing compounds such as methylguanidine and free arginine and its derivatives as well as with the arginyl side chain present on proteins.
  • the guanidino group of arginine may react with 3- deoxyglucosone (a reactive sugar, abbreviated 3DG, derived from the decomposition of the Amadori product; see Background) to form an advanced glycosylation endproduct.
  • 3DG reactive sugar
  • the present invention advantageously provides methods for preparing antibodies, and preferably monoclonal antibodies, having the binding characteristics of monoclonal antibody 4E1 by immunizing with an antigen such as 3DG-RNase, 3DG-lysozyme, 3DG-BSA and 3DG-KLH. Any such antigen may be used as an immunogen to generate antibodies with the immunological characteristics of monoclonal antibody 4E1.
  • antigen such as 3DG-RNase, 3DG-lysozyme, 3DG-BSA and 3DG-KLH.
  • Any such antigen may be used as an immunogen to generate antibodies with the immunological characteristics of monoclonal antibody 4E1.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • AGEs formed from 3DG represent an alternative and useful approach to the detection and quantitation of long-term glycosylation damage in the body integrating both time of exposure and levels of blood sugar in contact with the various susceptible organs, tissues, and protein masses in the body.
  • the antibodies of the present invention offer a different measure of such damage, i.e. , from non-oxidation-derived AGEs (supra), as well as means for evaluating the effectiveness of lifestyle changes and therapies directed at reducing glucose levels and eliminating AGEs from the body.
  • the monoclonal antibodies of the present invention share properties with previously-described anti-AGE antibodies in that they react with AGEs derived from glucose.
  • the antibodies of the present invention may be distinguished from that of the prior art by several means.
  • the instant antibodies do not detect AGEs derived from ribose or other 5-carbon sugars, nor do they detect pentosidine, carboxymethyllysine, nor the reaction product of 3DG with aminoguanidine or lysine, nor the reaction product of methylglyoxal with L- arginine.
  • Example 1 an ELISA using monoclonal antibody 4E1 on the fractions collected upon chromatographic separation of the various intermediates and products present in a reaction between 3DG and N e -benzoyl-L-arginine amide clearly distinguished the imidazolone S17 of Konishi et al. as cited above [amino-5-(2,3,4-trihydroxybutyl)- 4-imidazolone] , from the epitope to which the antibodies of the present invention bind.
  • the ALI compound (arginyl-lysyl-imidazole) described by Bucala et al. is derived from both arginine and lysine, and thus structurally differs from the 4E1 immunological epitope.
  • the guanidino group-derived AGEs detectable by the antibodies of the present invention arise from a non-oxidative pathway, in contrast to other AGEs such as carboxymethyllysine whose formation requires one or more oxidation steps.
  • the changes over time in the levels of immunological epitopes detectable in the body by the antibodies of the present invention reflect the rate of formation of guanidino group-derived AGEs minus their rate of removal or decomposition.
  • Various factors may cause an increase in their level, such as increased levels of blood glucose, a symptom of diabetes, and factors which interface with AGE metabolism and excretion, such as decreased AGE scavenger activity by macrophages and other cell types, and renal disease.
  • Factors which result in the decrease of guanidino group-derived AGEs include treatment with inhibitors of their formation, such as aminoguanidine, and agents that chemically cleave AGEs, both promoting a reduction in AGE burden in the body and addressing the attendant pathology.
  • monitoring guanidino group-derived AGE levels in samples of bodily fluids or tissue samples offers diagnostically-useful information for changes in the health of an individual as well as monitoring therapeutic intervention.
  • the antibodies of the present invention also detect an adduct formed from the reaction of 3DG with methylguanidine.
  • Methylguanidine is a metabolite of nitrogen breakdown in the body and the circulating level is elevated in patients with renal disease. Detection of this adduct may provide an indication of the level of 3DG and/or methylguanidine in a patient and thus the propensity to form toxic AGEs which cause pathology.
  • Sources of samples for the measurement of guanidino group-derived AGEs in accordance with the present invention may be chosen from the appropriate bodily fluids or tissue samples related to the condition to be detected, or treatment to be evaluated.
  • AGEs may be detected in plasma, serum and urine, on red blood cell membranes, on hemoglobin, and lipoprotein. Serum, plasma or urine may be used directly in an assay.
  • Hemoglobin may be isolated from whole blood by lysis of packed red blood cells.
  • Lipoprotein may be isolated prior to assay or guanidino group-derived AGEs detected thereon by first capturing lipoprotein with a specific antibody, e.g.
  • Tissue samples such as biopsy materials, may also be used as a source for the assay of guanidino group-derived AGEs.
  • Renal biopsy tissue for example, may be digested with collagenase or another suitable enzyme in order to solubilize proteins, after which the AGE assay may be performed.
  • the antigen may be conjugated to an immunogenic carrier, e.g. , bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), or the carrier may be reacted with 3- deoxyglucosone.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • 3-deoxyglucosone is derived from the decomposition of the Amadori product, which itself may be derived from the reaction of glucose with an amino group of a protein or other amine-containing molecule, glucose may be used to produce the immunogen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV -hybridoma technique to produce human monoclonal antibodies (Cole et al. , 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. , pp. 77-96).
  • monoclonal antibodies can be produced in germ-free animals utilizing technology set forth in PCT/US90/02545.
  • human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).
  • human hybridomas Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030
  • transforming human B cells with EBV virus in vitro Cold-et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96.
  • techniques developed for the production of "chimeric antibodies” or "humanized antibodies” (Morrison et al.
  • Chimeric antibodies are those that contain a human Fc portion and a murine (or other non-human) Fv portion; humanized antibodies are those in which the murine (or other non-human) complementarity determining regions (CDR) are incorporated in a human antibody; both chimeric and humanized antibodies are monoclonal.
  • Such human or humanized chimeric antibodies are preferred for use in in vivo diagnosis or therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogeneic antibodies to induce an immune response, in particular an allergic response.
  • Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • Such antibody fragments can be generated from any of the polyclonal or monoclonal antibodies of the invention; preferably, such antibody fragments are generated using monoclonal antibody 4E1.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. , radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, imrmmodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g.
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or other reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • such an antibody can be selected on the basis of an ability to compete for binding of monoclonal antibody 4E1 to such AGEs.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of AGE-modified proteins or tissues, e.g. , for Western blotting, ELISA, detecting AGE-modified tissue in situ, measuring levels of AGE- modified molecules, for instance including proteins, peptides, lipids and nucleic acids, and, in particular, hemoglobin-AGE, immunoglobulin-AGE, and LDL-AGE, in appropriate physiological samples, such as serum and urine samples.
  • the tissue and end organ damage caused by advanced glycosylation accumulates over a period of months to years. Diabetic complications progress over a similar duration, so that it is advantageous to detect earlier the AGE accumulation that has been linked to the development of pathology in such disease states.
  • the antibodies of the invention can be used to detect the presence of AGEs such as but not limited to, hemoglobin-AGE, albumin- AGE, lipid-AGEs, and AGE-modified peptides.
  • AGEs such as but not limited to, hemoglobin-AGE, albumin- AGE, lipid-AGEs, and AGE-modified peptides.
  • the presence of a disease or disorder associated with AGEs can be assessed by detecting higher levels of AGEs in a biological sample from a subject who suffers from such a disease or disorder, as compared to a normal individual.
  • the effectiveness of an agent, e.g. , aminoguanidine, to prevent or inhibit the formation of AGEs can be evaluated by observing a decrease in the level of AGEs in biological samples obtained from a subject over a time interval.
  • Hb-AGE hemoglobin-AGE
  • WO 93/13421 International Publication No. WO 93/13421
  • the present invention also extends to the measurement of other AGEs and particularly serum and urinary AGE-modified proteins and AGE-modified peptides.
  • Serum and urinary AGE-modified peptides like lipid-AGE and Hb-AGE, represent circulating markers of AGE accumulation that reflect the onset and extent of pathologies and other dysfunctions where such accumulation is a characteristic.
  • those AGE-related and diabetic conditions where increased levels of AGEs have been observed such as, for example, atherosclerosis, cataracts and diabetic nephropathy, may be monitored and assessed over the long term by the measurement of these AGEs, particularly by resort to the diagnostic methods disclosed herein.
  • serum peptide- AGEs can be used as an indicator that reflects glomerular filtration rate (GFR) and kidney damage.
  • Urinary peptide- AGEs may be used as an indicator to measure the turnover in tissue proteins, and more particularly, tissue protein bearing AGE modifications.
  • a blood sample is drawn and a separation procedure can be used.
  • a procedure such as that described in International Publication No. WO 93/13421 by Bucala et al. can be used.
  • hemoglobin-AGE the cellular blood components can be separated from the serum, and hemoglobin can be extracted from the red blood cells. The serum level of LDL-AGE, peptide-AGEs and the presence or extent of Hb-AGEs present can then be evaluated.
  • a broader time frame at which blood glucose levels become uncontrolled can be estimated, e.g., a 60 day range predictable by Hb-AGE for instance, extends the period to be assessed for glycemic control to before the 3-4 week time frame which is measured by Hb-A ]c determination.
  • the analyses of Hb-AGE and serum peptide-AGEs can be run together with a glucose level determination in blood or urine, a glucose tolerance test, and other tests useful for assessing diabetes control including the measurement of urinary peptide-AGEs, to give a complete patient profile.
  • LDL-AGEs are measured using a polyclonal or the preferred monoclonal antibody of the present invention against guanidino group-derived AGEs in combination with an anti-LDL antibody (such as, but not limited to, anti-ApoB).
  • an anti-LDL antibody such as, but not limited to, anti-ApoB.
  • Another aspect of the invention addresses advanced glycosylation endproducts which can be detected in the urine. Proteins, including peptides, are excreted in the urine in very low amounts in normal individuals, and at ever increasing levels as kidney function declines until levels decline in kidney failure. The presence and/or level of urinary peptide-AGEs reflective of the turnover of tissue AGEs can be determined, correlated to and predictive of particular diseases or conditions.
  • the presence of guanidino group-derived AGE-peptides in the urine may be a symptom of numerous diseases or conditions reflective of a net catabolic state as would exist when the host or patient is undergoing invasion as by infection. Under such circumstances, the host mobilizes against the invasive stimulus by the secretion of numerous factors such as cytokines that suspend the anabolic energy storage activity and cellular repair activities and promote instead the catabolic depletion of energy stores and the recruitment of leukocytes and other factors to fight and neutralize the stimulus.
  • the measurement of urinary peptide-AGEs provides yet another index of possible invasive activity in the host, such as cachexia and shock. Thus, one can measure the presence or level of peptide-AGEs in urine, and correlate this level to a standard.
  • the normal level may be low. In diabetic patients, the level of peptide-AGEs may be greater. Alternatively, in a subject suffering from AGE-associated advanced renal disease, the level of urinary peptides may be greatly decreased owing to the onset of renal failure. In patients experiencing infection or other trauma, the level of peptide-AGEs may be significantly greater than in normal individuals. Thus, the advancement or worsening of diabetes prior to the onset of renal complications, the onset of renal complications associated with diabetes or other AGE-related diseases, or the presence of infection could be detected by detecting urine levels of peptide-AGEs.
  • the anti-AGE antibodies of the invention can also be used in the treatment of patients to reduce the level or accelerate the removal of circulating AGEs or AGE- modified molecules, or similar such AGEs or AGE-modified molecules, which may be present in abnormally elevated levels in certain tissues, e.g. , pancreas, liver, kidney or brain, and which AGEs may be undesired. Treatment may be by administration to the individual, where a humanized monoclonal antibody is preferred.
  • Elevated levels of circulating guanidino group-derived AGEs may be removed by extracorporeal treatment whereby the individual's blood is cycled ex vivo through a device in which immobilized antibodies of the present invention bind and sequester molecules bearing guanidino group-derived AGEs from the blood.
  • the anti-AGE monoclonal antibody may be used to purify proteins that have been specially cultivated or produced for use as therapeutic agents.
  • the therapeutic use of such proteins is increasing in prominence and importance, and such exogenous proteins (like the host's own tissue and circulating proteins) are susceptible to glycation and the formation of AGEs.
  • Such AGEs are chemically reactive and biologically active, so it is desirable to limit their introduction into a host during therapy.
  • the present invention includes a method for purification of batches of such proteins by bringing them into contact with, for example, a quantity of the anti-AGE monoclonal antibody of the present invention or an antigen-binding fragment thereof, immobilized on a suitable substrate.
  • a suitable substrate could be refreshed or replaced periodically in the instance of a commercial process, so that a continuous circulation of protein material past the substrate and subsequent separation of the protein-AGE component could be conducted.
  • All of the protocols disclosed herein may be applied to the qualitative and quantitative determination of advanced glycosylation endproducts and to the concomitant diagnosis and surveillance of pathologies in which the accretion of advanced glycosylation endproducts is implicated.
  • Such conditions as diabetes and the conditions associated with aging, such as atherosclerosis and skin wrinkling represent non-limiting examples, and accordingly methods for diagnosing and monitoring these conditions are included within the scope of the present invention.
  • the present invention also includes assay and test kits for the qualitative and/or quantitative analysis of the extent of the presence of advanced glycosylation endproducts.
  • assay systems and test kits may comprise a labeled component prepared, e.g., by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the anti-AGE monoclonal antibody of the present invention or an antigen-binding fragment thereof, or to a binding partner thereof.
  • One of the components of the kits described herein is the anti-AGE monoclonal antibody of the present invention or the antigen-binding fragment thereof, in labeled or non-labeled form.
  • kits may be used to measure the presence of advanced glycosylation endproducts on recombinant or other purified proteins, and particularly those destined for therapeutic use, to assay them for AGE presence in a first instance, and in a second instance, to assist in their further purification free from material with undesired AGE modifications.
  • kits will contain at least the monoclonal antibody or an antigen-binding fragment thereof of the invention, means for detecting immunospecific binding of said antibody or fragment thereof to AGE components in a biological sample, and directions, of course, depending upon the method selected, e.g. , "competitive” , “sandwich” , “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • the preferred diagnostic test kit may further comprise a known amount of a binding partner to an anti-AGE antibody as described above, generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable label.
  • a test kit of the invention may also further comprise a second antibody, which may be labelled or may be provided for attachment to a solid support (or attached to a solid support).
  • a second antibody may be, for example, an anti-AGE antibody, or an antibody specific for the non-AGE portion of the analyte to be assessed for AGE modification, or an AGE-component. Examples of the latter include, but are not limited to, anti-hemoglobin, anti-albumin, and, as shown herein, anti-ApoB.
  • Such antibodies to the "carrier" portion of an AGE component can be polyclonal or monoclonal antibodies.
  • PBS phosphate-buffered saline
  • PBS may be prepared by dissolving 8.0 grams of NaCl, 0.2 grams of KC1, 1.44 grams of Na 2 HPO 4 , and 0.24 grams of KH 2 PO 4 in 800 ml of distilled water, adjusting the pH to 7.4, and the volume to 1 liter.
  • the resulting solution may be dispensed in convenient volumes and sterilized by autoclaving, and may be stored at room temperature.
  • the terms “Wash Solution” and “TBS-T Wash Solution” where present refer to the following: Tris Buffered Saline-Tween (TBS-T) (0.1 M Trizma, 0.15 M NaCl, 0.05 % Tween-20, 0.02% sodium azide, adjusted to pH 7.4 with HC1).
  • the term “Assay Buffer” refers to a solution of PBS containing 2% BSA, 0.2% Tween-20, and 0.2% sodium azide. The concentrations of the components comprising the Assay Buffer as may appear in the Examples listed below may vary within the scope of the present invention. Naturally the foregoing formulations are illustrative and may vary within the skill of the art, and are presented herein in fulfillment of the duty to present the best mode for the practice of the invention.
  • EXAMPLE 1 A HYBRIDOMA THAT SECRETES A GUANIDINO
  • KLH Keyhole limpet hemocyanin
  • BSA BSA was added at a concentration of 10 mg/ml to a solution of 3-deoxyglucosone (3DG, 0.1 M) in 100 mM sodium phosphate buffer, pH 7.4, and incubated at 50°C for 96 hours. After incubation, the KLH solution was dialyzed against PBS containing 0.1 m NaBH 4 for 1 hour and then against two changes of PBS for 4 hours each. The protein concentration of the 3DG-modified KLH was determined using a Lowry assay ans the sample was stored at -20°C.
  • mice used were female BALB/C approximately 8 weeks of age. Each mouse was immunized subcutaneously and intraperitoneally with 100 ⁇ l of a preparation containing 100 ⁇ g KLH-3DG in Ribi adjuvant. Mice were boosted subcutaneously and intraperitoneally at day 14 with the same preparation. On day 21, a test bleed was taken from the tail vein and serum prepared. The mouse showing the highest titer as determined in the antisera test bleed titering procedure described below was selected and boosted Intraperitoneally with 100 ⁇ g KLH-3DG in PBS and again on days 1 and 2 thereafter with 50 ⁇ g KLH-3DG in PBS. On day three, the spleen was removed. Antisera Test Bleed Titering
  • a dilution of 1/100 of each serum sample to be titered was prepared in PBS containing 0.1 % BSA for titer determination.
  • Pre-immune sera noted above were diluted in the same manner as the immune sera and used as controls.
  • Microtiter wells were coated with 1.0 ⁇ g of BSA-3DG antigen.
  • the antigen-coated wells were sealed with Mylar sealing tape (Corning) and incubated overnight at 4°C.
  • the microtiter plates were subsequently washed 6 times with TBS-T Wash Solution and blocked for one hour at 37 °C by adding 200 ul of a solution of PBS containing 0.2% BSA and 0.2% sodium azide.
  • microtiter plates were washed as before and 100 ul of the dilutions of pre-immune and immune sera were added. After incubation for 2 hrs. at room temperature, the microtiter plates were washed as described above and 100 ul of a goat anti-mouse IgG (gamma chain specific) alkaline-phosphatase-conjugated antibody (ICN) was added to all wells and incubated for 1 hr. at room temperature. The microtiter plates were washed as before and 100 ul of p-nitrophenyl phosphate substrate (Sigma) was added to all wells and incubated for 30 minutes at room temperature. After the incubation period, the plates were read at 410 nm on a microtiter plate reader.
  • ICN gamma chain specific alkaline-phosphatase-conjugated antibody
  • Hybridoma production was carried out by fusing the mouse spleen cells with the myeloma X63AG8.653 cell line as described elsewhere (Harlow, E. and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • Hybridoma Screening Procedure After fusion of spleen cells with the myeloma cell line, 100 ⁇ l of the 50 ml fusion mixture was added to each of 96 wells in 5 microwell cell culture plates (Corning). The plates were numbered 1 to 5, the rows of each plate by letter, and the columns by number to give a coding system that identified the parental cell cultures that developed from each drop of the fusion mixture. After culture in selection media described in Harlow and Lane, supra. , hybridoma cultures were screened for antibody production to AGE antigen as follows:
  • BSA-3DG-coated wells were prepared as described in the Antisera Test Bleed Titering section above.
  • the antigen-coated plates were used to screen cell culture supernates from each of the parental cultures.
  • the parental supernates were diluted 1:2 in PBS containing 0.2% BSA and 100 ⁇ l of each added to one well of a BSA- AGE coated microtiter plate.
  • the plates were incubated at room temperature for 2 hours and subsequently washed 6 times with TBS-T Wash Solution.
  • Hybridoma F52-4E1 which produces monoclonal antibody 4E1 was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville MD 20852, on March 31 , 1998, and assigned Accession Number HB- 12500.
  • Arginine, methylguanidine and guanidine were reacted with 3DG (0.2 M) in 200 mM sodium phosphate buffer, pH 7.4, for 24-72 hours at 37°C. These mixtures, along with control compounds at the same concentrations, were then tested by competitive ELISA (on AGE-BSA coated plates) using 4E1 as the primary antibody. Inhibition curves are least squares regression analysis of log/logit transformed data. The concentrations for 50% inhibition points are calculated from the linear regression analysis.
  • Direct ELISA and competition ELISA For direct ELISA, BSA-AGE or 3DG- modified BSA was coated on microtiter plates, the unbound sites were blocked by incubation with Assay Buffer. The plate was washed six times and increasing concentrations of MAb in Assay Buffer were added. After this incubation, the plate was again washed and incubated with alkaline-phosphatase labeled goat anti-mouse antibodies (ICN, Costa Mesa CA) diluted 1:2000 in Assay Buffer. The unbound antibodies were removed by extensive washing and the bound antibodies were detected by addition of p-nitrophenylphosphate in recording the optical density at 410 nm.
  • ICN alkaline-phosphatase labeled goat anti-mouse antibodies
  • the competition ELISA was performed by pre-coating microtiter plates with BSA- AGE and blocking with Assay Buffer. The plate was washed and niAb 4E1 and increasing concentrations of the competitors listed in Table 1 were added and simultaneously incubated for 1 hr at 22 °C. The unbound materials were removed by extensive washing and the bound n Ab was detected with alkaline phosphatase labeled anti-mouse antibodies similar to direct ELISA. All washes were in TBS-T wash solution; all incubations proceeded for 1 hr at 22 °C.
  • the elution program was 20% methanol in water for 20 min at a flow rate of 10 ml/min, followed by a gradient of 20 to 60% methanol in water over 40 minutes.
  • the column eluate was monitored by UV at 235 nm.
  • the fractions of interest eluted between 40 and 55 minutes.
  • the seven major peaks in this time period were collected and brought to dryness using a SpeedVac.
  • the dried samples were reconstituted in 1 ml water and the UV spectra taken. Using fraction 1 as a reference (1:30,000), all of the fractions were diluted so that an equivalent amount of material by UV was present.
  • These samples were then tested in the competitive ELISA (using AGE-BSA-coated plates) with 4E1 as the primary antibody.
  • the HPLC elution pattern by UV absorbance and the ELISA results for each of the seven fractions is shown in Figure 2 A and B.
  • fraction 4 contained the greatest amount of 4E1 antibody-reactive material, followed by fractions 3, 1, and 5. Little to no antibody reactive material was present in fraction 7.
  • the same reaction between 3DG and benzyl arginine was performed at 50°C as done by Konishi et al.(1994, Biosci. Biotech. Biochem. 58: 1953-1958), and a similar HPLC elution profile (Figure 3 A) to that seen previously was found.
  • an Inertsil 5 OBS-2 (1 cm x 25 cm) column (Metachem Technologies) was used with an elution program of 10-40% methanol in water in 30 minutes at a flow rate of 2.5 ml/min.

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Abstract

La présente invention a trait à des anticorps monoclonaux dirigés contre des produits finaux de glycosylation avancée dérivés de groupes guanadino, qui sont formés in vivo et croisés avec des produits finaux de glycosylation avancée formés in vitro, et à des méthodes diagnostiques et thérapeutiques basées sur ceux-ci. Plus particulièrement, l'invention a trait à un anticorps monoclonal ou à un fragment de liaison à l'antigène de cet anticorps, qui réagit avec des produits finaux de glycosylation avancée (AGE) produits in vivo. L'anticorps monoclonal ou le fragment de liaison à l'antigène de cet anticorps possède une caractéristique de liaison immunologique de l'anticorps monoclonal 4E1 produit par l'hybridome F52-4E1, déposé à l''American Type Culture Collection (ATCC)' le 31 mars 1998, et auquel a été attribué le numéro d'entrée HB-12500.
EP99928516A 1998-06-09 1999-06-09 Anticorps monoclonaux specifiques de produits finaux de glycosylation avancee derives de groupes guanidino dans des echantillons biologiques Withdrawn EP1086136A1 (fr)

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