EP0485476A1 - Antigenes specifiques aux plaques d'atherosclerose, anticorps diriges contre ces antigenes et utilisations de ces antigenes et de ces anticorps - Google Patents
Antigenes specifiques aux plaques d'atherosclerose, anticorps diriges contre ces antigenes et utilisations de ces antigenes et de ces anticorpsInfo
- Publication number
- EP0485476A1 EP0485476A1 EP90912266A EP90912266A EP0485476A1 EP 0485476 A1 EP0485476 A1 EP 0485476A1 EP 90912266 A EP90912266 A EP 90912266A EP 90912266 A EP90912266 A EP 90912266A EP 0485476 A1 EP0485476 A1 EP 0485476A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- antibody
- antigen
- reagent
- enzyme
- plaque
- 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.)
- Withdrawn
Links
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- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Definitions
- Atherosclerosis is the progressive narrowing of the lumen (inner passageway) of arterial blood vessels by layers of plague (fatty and fibrous tissues). Atherosclerosis can occur in any artery. In coronary arteries it may result in heart attacks; in cerebral arteries it may result in strokes; and in peripheral arteries it may result in gangrene of the legs and feet.
- Atherosclerosis is the single largest medical problem currently facing the United States and other developed countries. Approximately 40 million people in the United
- Heart attack and stroke represent the first and third leading causes of death in
- the endothelium is located between the blood and arterial tissue and serves as a barrier against the accumulation of blood components in the vascular wall. Formation of atherosclerotic lesions in the sub-endothelium is associated with major coronary artery disease and stroke. The causes and detection of such lesions have been intensely investigated.
- Endothelial injury is believed to be an initial step in the formation of the atherosclerotic lesions and may be caused by hemodynamic strain, hypercholesterolemia, hypertension and immune complex disease. Endothelial injury leads to cholesterol accumulation and intimal thickening, cellular proliferation, and formation of connective tissue fibers. IgG and complement factor C3 accumulation in injured endothelial cells and nonendothelialized intima has been observed. Hononuclear phagocytes derived from blood are also part of the cell population in atherosclerotic lesions. The mechanism of plague formation is not fully known. However, a probable mechanism is that fatty deposits lead to an influx of macrophages, which in turn are followed by T cells, B cells, and antibody production.
- soluble proteins have been extracted from human atherosclerotic plague, including IgA, IgG, IgM, B1C(C3), alpha 1 -antitrypsin, alpha 2 -macroglobulin, fibrinogen, albumin, LDL, HDL, alpha 1 -acid glycoprotein, B 2 -glycoprotein, transferrin and ceruloplasmin.
- the diseased intima was also found to contain a small amount of tissue-bound IgG, IgA and B1C [Hollander, W. et al.. Atherosclerosis, 34:391-405 (1979)].
- IgG has been determined in lesions and adjacent endothelial tissue [Parums, D.
- Atherosclerosis is generally a diffuse disease
- human coronary atherosclerosis lends itself to bypass procedures because the major site of plaque formation is usually proximally distributed.
- direct coronary artery bypass has become the most frequently selected form of myocardial revascularization.
- the aorta-coronary artery vein graft or the internal mammary artery graft have become technically standardized and have high long-term potency rates. These long-term results, however, can be compromised by progressive atherosclerosis distal to the graft anastomosis. Other cases are inoperable because of distal disease. Previously, distal lesions have been ignored or, in selected cases, treated by endarterectomy although neither approach has proved entirely satisfactory.
- Radioimaging thrombi with radiolabeled monoclonal antibodies to platelets was first described by Peters, A., et al. [British Medical Journal, 293:1525-1527 (December, 1986)].
- DTPA-coupled antibodies radiolabeled with metallic radionuclides has been described by Hnatowich, D., et al. [Journal of Immunological Methods, 65: 147-157 (1983)].
- NMRI NMR
- ultrasound and X-ray imaging with metal chelates are described in U.S Patent 4,647,447.
- antibody coupling of metal chelates is mentioned at column 7, line 42.
- Monoclonal antibodies labeled with polymeric paramagnetic chelates and their use in NMRI methods have also been described [Shreve, P. et al., Magnetic Resonance in Medicine, 3:336-340 (1986) and Brady, T. et al. in Proceedings of the Society of Magnetic Resonance in Medicine, Second Annual Meeting, Soc. of Magnetic Resonance in Medicine, Inc., San Francisco, p. 10, (1983), referenced by Koutcher, J. et al., J. Nucl. Med., 25:506-513 (1984)].
- GLA gamma-carboxygluteuaic acid
- GLA specific antibodies bind with GLA present in advanced atherosclerotic plaque having calcium deposits.
- Lian et al. report that GLA is not found in uncalcified plaques and that GLA is found in cardiac valves and aortas, and in circulating proteins such as prothombin, clotting factors VII, IX and X, Protein C and Protein S.
- the GLA binding antibodies of Lian et al. do not selectively bind to atherosclerotic plaque.
- the atherosclerotic plaque antibodies of the subject invention bind to all stages of atherosclerotic plaque including non-calcified stages, and do not selectively bind with GLA.
- the concept of plaque enhancement by application of a stain has been reported [Spears, J.
- the above-identified stains were selected for their ability to bind to components of the atherosclerotic plaque.
- the stain absorbs laser light concentrating the light at the stained surface. Some staining of healthy tissue occurs causing stain associated damage to the surrounding tissue. Because laser wavelength is limited to the absorption wavelength of the stain, chromophores offering optimum absorption of laser must be used to provide most controlled ablation.
- lasers have been used increasingly in microsurgery, both as scalpels and as coagulating instruments. Because of their ability to produce relatively bloodless incisions of great precision, as well as focal coagulation, they have been particularly useful in microsurgical procedures in the eye, central nervous system. nasal passages, cervix, gastrointestinal tract, skin, muscle, and even in small vessels.
- the subject invention provides an inexpensive, accurate method for determining the presence of atherosclerotic plaque both in vitro and in vivo.
- the subject invention provides methods of treating persons having atherosclerotic plaque which include enzyme treatment, and laser treatment.
- the subject invention provides a method of drug delivery to areas of atherosclerotic plaque.
- the subject invention provides a purified antigen indicative of the presence of atherosclerotic plaque characterized as having a complex carbohydrate structure having a molecular weight greater than 200,000 daltons and being present as an extracellular component of atherosclerotic plaque.
- the subject invention also provides a purified antigen wherein the antigen is characterized by its selective binding to the monoclonal antibody produced by hybridoma Q10E7. These antigens are characterized by existing in amounts which vary with the progression of atherosclerosis.
- the subject invention further provides antibodies to these antigens and methods of detecting the presence of both the antigens and the antibodies thereto. Methods for treating atherosclerosis are also provided.
- the subject invention also provides a method for reducing the amount of atherosclerotic plague in a blood vessel which comprises: a) contacting the atherosclerotic plaque with a reagent which is capable of specifically binding to both the plaque and to a proenzyme, the substrate of which enzyme is a connective tissue present in atherosclerotic plaque which, when cleaved, is capable of dissolving a component of the plaque under conditions such that the reagent binds to the plague so as to form a reagent-plaque complex; b) contacting the reagent-plaque complex with the proenzyme to which the reagent specifically binds under conditions such that the proenzyme is bound to the reagent forming a proenzyme-reagent-plaque complex; and c) contacting the proenzyme-reagent-plaque complex with an agent which is capable of specifically cleaving the proenzyme so that the proenzyme is converted into the enzyme under conditions such that the enzyme digests the plaque
- the subject invention further provides a method for diagnostic analysis comprising the steps of: a) obtaining a value for the body mass index (BMI) of a patient; b) obtaining a value for the concentration of an antigen or other serum or plasma analytes associated with a pathological condition or an antibody which binds with the antigen; c) plotting the body mass index of the patient against the antigen or antibody concentration of the same patient; and d) comparing the resulting value against a set of reference values to determine whether the resulting value exceeds the reference value and thereby indicates the presence of a pathological condition.
- BMI body mass index
- Figure 1 Pathways used for developing antibodies to the various stages of the atherosclerotic plaque specific antigen and for testing the antigen and antibodies made thereto.
- the dotted line represents the amount of binding of CAD serum compared to normal serum in each fraction.
- the solid line represents the amount of protein in each fraction as detected by absorbance at OD 280 .
- the dashed line represents a NaCl gradient from 0 to
- FIG. 3 DEAE fractionation (Analytical) - Antigen capture assay.
- the dotted line represents the amount of auto-antigen in each fraction as detected by binding to the monoclonal antibody produced by hybridoma 15H5. The results shown are for peroxidase conjugated antibody to the antigen and the plates are read at OD 450 .
- the solid line represents the amount of protein in each fraction as detected by absorbance at OD 280 .
- the dashed line represents a NaCl gradient from 0 to 1.0 M NaCl.
- Figure 4 Atherosclerotic plaque 15H5-antigen sizing.
- a mixture of the autoantigen and 4 size markers [Thyroglobulin (a); IgG (b); Ovalbumin (c); and Myoglobin (d)] were run through a BioSil analytical TSK-400 column.
- the auto-antigen was detected by binding with peroxidase-labeled 17H3 and 15H5. Binding is determined by measuring absorbance at OD 450 .
- Figure 5 Level of IgA which specifically binds to atherosclerotic plague antigen for persons with CAD, normal persons less than 35 years of age, and normal persons greater than 35 years of age.
- Figure 6 Levels of atherosclerotic plaque antigen as determinined by radioimmunoassay for persons with CAD, normal persons less than 35 years of age, and normal persons greater than 35 years of age.
- Figure 7 Atherosclerotic plaque antigen level for normal persons vs. age.
- Figure 8 Atherosclerotic plaque antigen level for persons with severe CAD, i.e. greater than 50% occlusion, vs. age.
- Figure 9 Atherosclerotic plaque antigen levels for persons with mild CAD vs. age.
- Figure 10 Level of IgG which specifically binds to atherosclerotic plaque antigen for normal persons vs. age.
- Figure 11 Level of IgG which specifically binds to atherosclerotic plaque antigen for persons with severe CAD, i.e. greater than 50% occlusion, vs. age.
- Figure 12 Level of IgG which specifically binds to atherosclerotic plague antigen for persons with mild CAD vs. age.
- Figure 13 Level of IgA which specifically binds to atherosclerotic plaque antigen for normal persons vs. age.
- Figure 14 Level of IgA which specifically binds to atherosclerotic plaque antigen for persons with severe CAD, i.e. greater than 50% occlusion, vs. age.
- Figure 15 Level of IgA which specifically binds to atherosclerotic plaque antigen for persons with mild CAD vs. age.
- Figure 16 Positive prevalence of atherosclerotic antigen, i.e. percent of persons above normal, vs. age group.
- Figure 17 Positive prevalence of antibody which specifically binds to atherosclerotic plaque antigen, for various age groups. Solid bars represent IgG. Cross-hatched bars represent IgA.
- Figure 18 Positive prevalence of either, or both, IgG or IgA which specifically binds to atherosclerotic plaque antigen for various age groups.
- Figure 19 Positive prevalence of either antibody (IgG or IgA) or antigen for various age groups.
- Figure 20 - A chromatographic blank run with just distilled water using a Dionex instrument for monosaccharides analysis with a CPPA-1 column.
- Figure 21 Chromatographic run of seven standard monosaccharides using a Dionex instrument for monosaccharides analysis with a CPPA-1 column, 15 mM NaOH in purified water.
- Figure 22 Chromatographic blank run with the auto- antigen affinity purification with the 15H5 monoclonal antibody using a Dionex instrument for monosaccharides analysis with a CPPA-1 column, 15 mM NaOH in purified water.
- IgG+A Values in each compartment are marked at the 98th percentile, so that 98 percent of subjects in such compartment are below the marked threshold. If a subject is above the threshold, such subject may be predisposed to atherosclerosis.
- FIG. 24 Graph of body mass index (BMI) against antigen. Values in each compartment are marked at the 100th percentile, so that 100 percent of subjects in such compartment are below the marked threshold. If a subject is above the threshold, such subject may be predisposed to atherosclerosis.
- BMI body mass index
- Figure 25 Antigen binding inhibitions percent inhibition of binding for antibodies produced by hybridoma 15H5 and 17H3 shown after pretreatment of antigen covered microbes with various dilutions of CAD serum.
- Figure 26 Antigen binding inhibition percent inhibition of binding of CAD serum after pretreatment of antigen covered microwells with various amounts of the antibodies produced by hybridoma 15H5 and 17H3.
- FIG. 27 Schematic representation of method No. 1 for purifying the forms of the atherosclerotic plaque antigen recognized by the antibodies produced by hybridomas Z2D3 and Q10E7.
- the peak representing the antigen form which binds to Z2D3 is determined by the ELISA method using the antibody produced by hybridoma Z2D3.
- Figure 30 Schematic of enzymatic reduction of atherosclerotic plaque by proenzyme targeting with plaque specific antibody fragments.
- C Representation of the enzyme including the propeptide portion which inhibits enzyme activity.
- D Representation of the antigen-bifunctional antibody proenzyme complex.
- Figure 32 Immunostaining of an unfixed 5 ⁇ thick frozen tissue section of human coronary artery, from a patient with advanced atherosclerosis using the ABC immunoperoxidase method, and counter stained with hematoxylin. Magnification is 20x.
- Figure 33 Immunostaining of an unfixed 5 ⁇ thick frozen tissue section of human coronary artery from a patient with early atherosclerosis using the ABC peroxidase method, and counterstained with hematoxylin. Magnification is 20X.
- Figure 34 Immunostaining of an unfixed 5 ⁇ thick frozen tissue section of human coronary artery from a patient with atherosclerosis, using the ABC peroxidase method, counterstained with hematoxylin. Magnification is 20X.
- Figure 35 Comparison of a section of rabbit aorta from the thorax region which was untreated, a, and a section of aorta from the same rabbit which was denuded so as to produce atherosclerotic plaque, b. Prior to sacrificing the rabbits, the rabbits were injected via the eeu: vein with 1 11 In-DTPA-Z2D3 monoclonal antibodies.
- the subject invention a purified antigen indicative of the presence of atherosclerotic plaque characterized as having a complex carbohydrate structure and a molecular weight greater than 200,000 daltons and as being present as an extracellular component of atherosclerotic plaque.
- This antigen is characterized by being synthesized by, or present in, smooth muscle cells. It has been purified and is characterized by its selective binding to the monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No. HB9839). This antigen is further characterized by being neutral in charge. This antigen is also characterized by having a carbohydrate profile depicted in Figure 22. It has been determined that the 15H5 antigen selectively binds to lectins.
- this can be further characterized by binding to the lectins Conavalia ensiformis, Triticum vulgaris, Lens culinaris, Ricinus commonis, and Triticum vulgaris, and by not binding to the lectins Arachis hypgaea, Bandeiraea simplicitolia. Diolichos biflorus, Glycine Max,
- Limulus polyphenus Phaseolus vulgaris-E, Phaseolus vulgaris-L, Pisum sativum. Sophova japonica, Ulex eurooaes, Ulex europaeus, and Vicia villosa.
- the 15H5 antigen is further characterized as being resistant to degradation by proteinases, deoxyribonucleases. Upases, and ribonucleases, while being partially susceptible to degradation by ⁇ -amylase, ⁇ -amylase, and glucoamylase.
- the antigen was evaluated for binding to antibodies which specifically bind to known components of atherosclerotic plague.
- the antigen may be further characterized as being non-reactive with antibodies which bind to apolipoproteins, human collagen, fibronectin, keratin, laminin, tenascin, and vitronectin.
- antigen characteristics include being reactive with the monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No. HB9840) after the antigen has been boiled for one hour, and being reactive with the monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No.
- the subject invention also provides an antigen or epitope associated with atherosclerosis and some normal tissue, characterized by its selective binding to the monoclonal antibody produced by hybridoma 17H3 (ATCC Accession No. HB 10189).
- antigen is provided by the subject invention, which antigen is characterized by being synthesized by, or present in, atherosclerotic plaque connective tissue and plaque smooth muscle cells is the antigen characterized as being a lipid-containing molecule which selectively binds to the monoclonal antibody produced by hybridoma Z2D3 (ATCC Accession No. HB 9840) or by hybridoma Z2D3/3E5 (ATCC Accession No. HB 10485). This antigen may be further characterized by having its ability to be used for histological staining destroyed upon treatment with acetone. Also provided are antigens indicative of the presence of normal smooth muscle cells.
- One such antigen is characterized by its selective binding to the monoclonal antibody produced by hybridoma Q10E7 (ATCC Accession No. HB 10188). The molecular weight of this antigen is greater than 150,000 daltons. This antigen is further characterized by being synthesized by, or present in, normal smooth muscle cells and normal connective tissue surrounding arteries.
- an above-described antigen is labeled with a detectable marker.
- This marker may be any marker known to one skilled in the art.
- the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.
- the preferred marker is an enzyme, preferably horseradish peroxidase or alkaline phophatase.
- the subject invention also provides purified antibodies which specifically bind to an atheroclerotic plaque antigen or to an antigen associated with normal smooth muscle cells and connective tissue.
- the antibody is labeled with a detectable marker.
- the choice of marker used will vary depending upon the application. However, the choice of marker is readily determinable to one skilled in the art.
- the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.
- These labeled antibodies may be used in immunoassays as well as in histological applications to detect the presence of atherosclerotic plaque.
- the marker is an enzyme, and it is most preferred that the enzyme is horseradish peroxidase or alkaline phosphatase.
- the above-identified antibodies may be either polycional or monoclonal, with the monoclonal antibody being a preferred embodiment.
- This invention provides monoclonal antibodies directed to atherosclerotic plaque antigens which include the monoclonal antibody produced by hybridoma 15H5 (ATCC Accession No. HB9839); the monoclonal antibody produced by hybridoma Z2D3 (ATCC Accession No. HB9840) and Z2D3/3E5 (ATCC Accession No.
- HB 10485 an IgG, which is a class switch variant of Z2D3, which is an IgM, as well as other daughter cell lines of Z2D3 such as Z2D3/5C5 (an IgG); and the monoclonal antibody produced by hybridoma 17H3 (ATCC Accession No. HB 10189).
- the monoclonal antibody produced by hybridoma Q10E7 (ATCC Accession No. HB 10188) is directed toward normal artery.
- Hybridomas 15H5, Z2D3, Z2D3/3E5, 17H3 and Q10E7 were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. HB 9839, HB 9480, HB 10485, HB 10189, and HB 10188, respectively.
- the invention provides a recombinant polypeptide which comprises an amino acid sequence which is substantially the same as the amino acid sequence of the hypervariable region of monoclonal antibody Z2D3, Z2D3/3E5 and other daughter cells lines of Z2D3 or Q10E7.
- a polypeptide may also obtain such a polypeptide by nonrecombinant methods, such as for example, proteolytic digestion.
- a chimeric antibody or a fragment thereof comprising such a recombinant polypeptide is also provided, particularly a chimeric antibody comprising the amino acid sequences of a human framework region and of a constant region from a human antibody so as to "humanize” or render nonimmunogenic the hypervariable region of the mouse Z2D3, Z2D3/3E5 or Q10E7.
- the polypeptide or chimeric antibody or fragment derived by site-directed mutagenesis, especially site-directed mutagenesis which confers equivalent or better binding properties.
- the fragments of the chimeric antibody include Fab, F(ab) 2 , F v and V H fragments.
- the subject invention also provides for an atherosclerotic plaque antigen bound to a solid support and an antibody which specifically binds to an atherosclerotic plague antigen bound to a solid support.
- the monoclonal antibody produced by hybridoma 15H5 is bound to a solid support.
- Anti-plaque antibody or the atherosclerotic plaque antigen may be bound to an insoluble support by conventional processes. Procedures for binding of antibodies to insoluble supports are described in U.S. Patents 3,551,555, 3,553,310, 4,048,298 and RE-29,474, for example. Binding of antibodies to polystyrene by adsorption has been described in U.S. Patents 3,646,346 and 4,092,408, for example. Binding of protein containing antigens to a variety of insoluble supports has been described in U.S. Patent 3,720,760.
- a variety of materials may be used as the insoluble support, the primary consideration being the binding characteristics of the anti-plaque antibody or the plaque antigen to the surface, the absence of interference with the anti-plaque antibody and plaque antigen conjugating reaction or with other reactions which may be employed to determine the presence and extent of the conjugating reaction.
- Organic and inorganic polymers both natural and synthetic, can be used as the insoluble support. Examples of suitable polymers include polyethylene, polypropylene, polybutylene.
- vinyl polymers such as polyvinyl acetate, polyvinyl chloride, polyvinylidene chloride, polyvinyl fluoride, and the like
- insoluble support can the latexes of the above polymers, silica gel, silicon wafers, glass, paper, insoluble protein, metals, metalloids, metal oxides, magnetic materials, semi-conductive materials, cermets and the like.
- substances which form gels such as proteins such as gelatins, lipopolysaccharides, silicates, agarose, polyacrylamides or polymers which form several aqueous phases such as dextrans, polyalkylene glycols (alkylene with 2 to 3 carbon atoms) or surfactants, e.g. amphophilic compounds such as pohospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like.
- One diagnostic support comprises polystyerene, styrene copolymers, or polyolefins such as polyethylene or polypropylene, and acrylate and methacrylate polymers and copolymers.
- the anti-plaque reagent antibody or the plaque antigen can be bound to the insoluble support by adsorption, ionic bonding, van der Waals adsorption, electrostatic bonding, or other non-covalent bonding, or it can be bound to the insoluble support by covalent bonding.
- a particularly advantageous support for this procedure comprises a microtiter plate having a plurality of wells.
- the well surface or plastic cup inserts therein can constitute the antigen or antibody support. If the determination will require the use of fluorometric measurements, the microtiter plate or the well inserts are advantageously opaque to light so that excitation light applied to a well does not reach or influence contents of the surrounding wells.
- the well can be coated with a layer having free isocyanate groups such as a polyether isocyanate, and application of the antibody or antigen in aqueous solution thereto effects the requisite bonding.
- the antibody or antigen can be coupled to a hydroxylated material by means of cyanogen bromide as described in U.S. Patent 3,720,760.
- the subject invention also provides a method for detecting in a biological sample an antigen present in, and indicative of the presence of, atherosclerotic plaque which comprises contacting the biological fluid with the antibody which specifically binds to the atherosclerotic plaque antigen under conditions such that the antibody binds to the antigen to form a detectable complex, detecting the complex so formed and thereby detecting any antigen in the biological sample.
- the biological sample is a tissue sample.
- Tissue samples may be used in a variety of histological techniques including but not limited to those illustrated throughout the application.
- the biological sample is a biological fluid. It is preferred that the biological fluid comprises blood, plasma, or serum. However, in the more preferred embodiment the biological fluid is serum.
- the antibody which binds specifically to the atherosclerotic plaque antigen is labeled with a detectable marker. The choice of marker is readily determinable to one skilled in the art.
- the antibody is a monoclonal antibody and more preferably the monoclonal antibody is produced by hybridoma 15H5 (ATCC Accession No. HB9839).
- the antibody be bound to a solid support.
- One preferred solid support is a bead formed of an inert polymer and another is a microwell.
- the subject invention also provides a method for quantitatively determining in a sample of a biological fluid the concentration of an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises: a) contacting a solid support with an excess of an antibody which binds specifically to an atherosclerotic plaque antigen under conditions permitting the antibody to attach to the surface of the solid support; b) removing unbound antibody; c) contacting the resulting solid support to which the antibody is bound with the sample of the biological fluid under conditions such that any antigen present in the sample binds to the bound antibody and forms a complex therewith; d) removing any antigen which is not bound to the complex; e) contacting any complex so formed with an excess of a detectable reagent which specifically binds to any antigen present in the complex so as to form a second complex which includes the antibody, the antigen, and the detecte ⁇ le reagent; f) removing any detectable reagent which is not bound in the second complex; g) quantitative
- the biological fluid comprises blood, plasma, or serum. More preferably, the biological fluid is serum.
- the solid support is a bead formed of an inert polymer and in another the solid support is a micrcwell.
- the reagent is labeled with a detectable marker, the choice of marker being determinable by one skilled in the art.
- the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.
- the reagent is an antibody labeled with a detectable marker.
- the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker is an enzyme, particularly effective enzymes being horseradish perosidase alkaline phosphatase.
- the subject invention also provides for the above method wherein the detectable reagent is labeled with an enzyme and step (g) comprises contacting the second complex with a specific substrate for the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.
- Another provision of the subject invention is a method for detecting in a biological sample an antibody which specifically forms a complex with an antigen present in, and indicative of the presence of, atherosclerotic plaque which comprises contacting the biological seunple with an atherosclerotic plaque antigen under conditions such that the antigen binds to the antibody in the biological seunple and detecting the antigen bound to the antibody and thereby detecting the antibody in the biological sample.
- the biological sample is a tissue sample.
- Tissue samples may be used in any histological technique known to one skilled in the art to detect and quantify the amount of antibody in the sample. The methods include, but are not limited to, the illustrations provided throughout the application.
- the biological sample is a biological fluid.
- the biological fluid comprises blood, plasma, or serum. More preferably the biological fluid is serum.
- the antigen is labeled with a detectable marker.
- Another embodiment of the invention is wherein the antigen is bound to a solid support. This allows the complex to be readily separated from the biological fluid and be detected.
- solid support is a bead formed of an inert polymer, and another is wherein the solid support is a microwell.
- the subject invention also provides a method for quantitatively determining in a sample of a biological fluid the concentration of an antibody which specifically forms a complex with an antigen present in, and indicative of the presence of, atherosclerotic plaque which comprises: a) contacting a solid support with an excess of an atherosclerotic plaque antigen under conditions permitting the antigen to attach to the surface of the solid support; b) removing unbound antigen; c) contacting the resulting solid support to which the antigen is bound with the seunple of the biological fluid under conditions such that any antibody present in the sample binds to the bound antigen and forms a complex therewith; d) removing any antibody which is not bound to the complex; e) contacting any complex so formed with an excess of a detectable reagent which specifically binds to any antibody present in the complex so as to form a second complex which includes the antigen, the antibody, and the detectable reagent; f) removing any detectable reagent which is not bound in the second complex; g) quantitative
- the biological fluid comprises blood, plasma, or serum. More preferably, the biological fluid is serum.
- the reagent is labeled with a detectable marker.
- the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker is an enzyme. The most preferred embodiment is when the enzyme is horseradish peroxidase or alkaline phosphatase.
- a further embodiment of the above-described method is wherein the detectable reagent is labeled with an enzyme and step (g) comprises contacting the second complex with specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.
- the subject invention discloses a method for quantitatively determining in a sample of a biological fluid the concentration of an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises: a) contacting a solid support with a predetermined amount of an antibody which binds specifically to atherosclerotic plague under conditions permitting the antibody to attach to the surface of the support; b) removing unbound antibody; c) contacting the resulting solid support to which the antibody is bound with a predetermined amount of antigen labeled with a detectable marker and with a sample of the biological fluid under conditions such that the antigen binds to the antibody bound to the solid support and forms a complex therewith; d) removing labeled antigen which is not bound to the complex
- One preferred solid support is a bead formed of an inert polymer, and another is wherein the solid support is a microwell.
- the detectable marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope.
- the marker is an enzyme, and more preferably the enzyme is horseradish peroxidase or alkaline phosphatase.
- the antigen is labeled with an enzyme and step (e) comprises contacting the labeled antigen bound to the solid support with specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.
- the subject invention further provides a method for quantitatively determining in a sample of a biological fluid the concentration of an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises: a) contacting a solid support with a predetermined amount of an antibody which specifically binds to an atherosclerotic plaque antigen under conditions permitting the antibody to attach to the surface of the support; b) removing any antibody not bound to the support; c) contacting the solid support to which the antibody is bound with the sample of the biological fluid under conditions such that any antigen present in the sample binds to the bound antibody and forms a complex therewith; d) removing any antigen which is not bound to the complex; e) contacting the complex so formed with a predetermined amount of plaque antigen labeled with a detectable marker under conditions such that the labeled antigen competes with the antigen from the biological fluid for binding to the antibody; f) quantitatively determining the concentration of labeled antigen not bound to the solid support; and g)
- the biological fluid comprises blood, plasma, or serum.
- the biological fluid is serum.
- the solid support is a bead formed of an inert polymer, and in another, the solid support is a microwell.
- the choice of marker is readily determined by one skilled in the art.
- the marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker is an enzyme. Although many enzymes produce a detectable product, preferred enzymes are horseradish peroxidase and alkaline phosphatase.
- step (f) comprises removing the labeled antigen which was not bound to the solid support and contacting it with a specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.
- Another method provided for the subject invention is a method for quantitatively determining in a sample of a biological fluid the concentration of an antibody which specifically forms a complex with an antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises: a) contacting a solid support with a predetermined amount of an atherosclerotic plague antigen under conditions permitting the antigen to attach to the surface of the support; b) removing unbound antigen; c) contacting the resulting solid support to which the antigen is bound with a predetermined amount of an antibody labeled with a detectable marker and with the sample of biological fluid under conditions such that the antibody binds to the antigen bound to the solid support and forms a complex therewith; d) removing any antibody which is not bound to the complex; e) quantitatively determining the concentration of labeled antibody bound to the solid support; and f) thereby quantitatively determining the concentration of antibody in the biological fluid.
- the biological fluid comprises blood, plasma, or serum. More preferably, the biological fluid is serum.
- the solid support is a bead formed of an inert polymer, and in another, the solid support is a microwell.
- detectable marker may be readily determined by one skilled in the art. It is preferred, however, that the detectable marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker is an enzyme, and most preferably the enzyme is horseradish peroxidase or alkaline phosphatase.
- the antibody is labeled with an enzyme and step(e) comprises contacting the labeled antibody which was displaced from the solid support with a specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.
- the subject invention further discloses a method for quantitatively determining in a sample of a biological fluid the concentration of antibody which specifically forms a complex with an atherosclerotic antigen which is present in, and indicative of the presence of, atherosclerotic plaque which comprises: a) contacting a solid support with a predetermined amount of an atherosclerotic plaque antigen under conditions permitting the antigen to attach to the surface of the support; b) removing any antigen which is not bound to the support; c) contacting the solid support to which the antigen is bound with the sample of the biological fluid under conditions such that any antibody present in the sample binds to the bound antigen and forms a complex therewith; d) removing any antibody which is not bound to the complex; e) contacting the complex so formed with a predetermined amount of a plaque antibody labeled with a detectable marker under conditions such that the labeled antibody competes with the antibody in the biological fluid for binding to the antigen; f) quantitatively determining the concentration of labeled antibody not
- the biological fluid comprises blood, plasma, or serum.
- the preferred biological fluid is serum.
- the choice of solid support may be readily determined by one skilled in the art.
- the solid support is a bead formed of an inert polymer, in another the solid support is a microwell.
- the markers used in the above-described method are a matter of choice to one skilled in the art. It is preferred that the detectable marker is an enzyme, a paramagnetic ion, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope. More preferably, the marker is an enzyme, and most preferably, the enzyme is horseradish peroxidase or alkaline phosphatase.
- a further embodiment of this method is wherein the antibody is labeled with an enzyme and step (f) comprises removing the labeled antigen which was not bound to the solid support and contacting it with specific substrate to the enzyme under conditions such that the enzyme reacts with the substrate to form a detectable product.
- the subject invention also provides a method for monitoring the progression of atherosclerosis which comprises determining the amount of atherosclerotic plaque antigen present in a sample of biological fluid of patient, and comparing the amount determined with the amount determined at earlier points of time, any change in the amount of antigen indicating a change in the extent of atherosclerotic plaque.
- Another provision of this invention is for a method for monitoring the efficacy of treatment of atherosclerosis which comprises determining the amount of the atherosclerotic plague antigen present in a sample of a biological fluid of a patient and comparing the amount determined at earlier points in time with a change in the amount of antigen indicating a change in the extent of atherosclerotic plague.
- a reagent for use in imaging atherosclerotic plaque which comprises an antibody which binds specifically to atherosclerotic plaque antigen labeled with a detectable marker.
- This invention also provides a composition comprising an amount of this reagent and a physiologically acceptable carrier.
- the detectable marker used is a matter of choice to one skilled in the art. It is preferred that the marker be a radioactive isotope, an element which is opaque to X-rays, a paramagnetic ion, or a chelate of a paramagnetic ion.
- Radioactive isotopes are commonly used in medicine and are well known to those skilled in the art. It is presently preferred that the marker be 1-123, 1-125, 1-128, 1-131, or a chelated metal ion of chromium-51, cobalt-57, gallium-67, indium-111, indium-113m, mercury-197, selenium-75, thalium-201, technetium-99m, lead-203, strontium-85, strontium-87, gallium-68, samarium-153, europium-171, ytterbium-169, zinc-62, rhenium-188, or mixtures thereof.
- the marker is technetium, iodine, indium or a metal ion chelate thereto.
- the marker is a paramagnetic ion.
- Paramagnetic ions are also commonly used in medicine. Examples of such markers included chelated metal ion of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), praseodymium (III), neodyminum (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), ytterbium (III), or mixtures thereof.
- the subject invention also provides a method for imaging atherosclerotic plaque which comprises contacting the atherosclerotic plaque to be imaged with a reagent which binds specifically to the atherosclerotic plaque antigen described above, under conditions such that the reagent binds to the atherosclerotic plaque and detecting the reagent bound thereto, thereby imaging the atherosclerotic plaque.
- Also provided is a method for imaging atherosclerotic plaque and adjacent normal tissue which comprises contacting the normal lumen to be imaged with an antibody which specifically binds to normal intima and/or media and which is labeled with a detectable marker; contacting the atherosclerotic plaque with a reagent described above under conditions such that the reagent binds to the atherosclerotic plaque; and detecting the reagents bound to the atherosclerotic plaque and adjacent normal tissue, thereby imaging the atherosclerotic plaque and adjacent normal tissue.
- the antibody which specifically binds to normal intima and/or media is a purified antibody which specifically binds to an antigen characterized by being synthesized by, or present in, normal smooth muscle cells and normal connective tissue surrounding arteries.
- the antibody is a monoclonal antibody produced by hybridoma Q10E7 (ATCC Accession No. HB 10188).
- the subject invention provides reagents for use in the method described above for imaging normal intima and/or media comprising an antibody labeled with a detectable marker as well as a composition comprising an effective imaging amount of such reagents and a physiologically acceptable carrier.
- the detectable marker used is a matter of choice to one skilled in the art. It is preferred that the marker be a radioactive isotope, an element which is opaque to X-rays, a paramagnetic ion, or a chelate of a paramagnetic ion. Markers that may be used in imaging normal tissue correspond to those described above for imaging atherosclerotic plaque.
- Another provision of the subject invention is a method for monitoring the progression of atherosclerosis which comprises determining the amount of an atherosclerotic plaque specific antigen present in a patient's blood vessels and comparing the amount determined with the amount determined at earlier points in time, a change in the amount of antigen indicating a change in the extent of atherosclerotic plaque.
- a method for monitoring the efficacy of treatment of atherosclerosis which comprises determining the amount of an atherosclerotic plaque specific antigen present in a patient's blood vessels and comparing the amount determined with the amount determined at earlier points in time, a change in the amount of antigen indicating a change in the extent of atherosclerotic plaque.
- Also provided for is a method for imaging atherosclerotic plaque in a subject which comprises: a) contacting the blood vessel walls containing atherosclerotic plaque with the above-described reagent for imaging plaque; b) detecting the reagent bound to the atherosclerotic plaque; and c) imaging the atherosclerotic plaque.
- a method for imaging atherosclerotic plaque and adjacent normal tissue in a subject which comprises contacting the normal lumen to be imaged with an antibody which specifically binds to normal intima and/or media and which is labeled with a detectable marker; contacting the blood vessel walls containing atherosclerotic plaque and surrounding area to be imaged with the reagent of claim 118 under conditions such that the reagent binds to the atherosclerotic plaque; and detecting the reagents bound to the atherosclerotic plaque and adjacent normal tissue, thereby imaging the atherosclerotic plaque and adjacent normal tissue.
- the antibody which specifically binds to normal intima and/or media is a monoclonal antibody produced by hybridoma Q10E7 (ATCC Accession No. HB 10188).
- Imaging may be done through any of the methods known to one skilled in the art. These methods include but are not limited to X-ray, CAT scan, PET scan, NMRI, and fluoroscopy.
- the subject invention provides a reagent for use in digesting atherosclerotic plaque which comprises en antibody which binds specifically to atherosclerotic plaque bound to an enzyme capable of digesting a component of atherosclerotic plaque.
- One such reagent comprises the monoclonal antibody produced by hybridoma Z2D3 or Z2D3/3E5 or other daughter cell lines and another comprises the 15H5 or 17H3 monoclonal antibody.
- Another such reagent comprises the chimeric antibody described above or a fragment thereof comprising the recombinant polypeptide which comprises an amino acid sequence which is substantially the same as the amino acid sequence of the hypervariable region of the monoclonal antibody produced by hybridoma Z2D3 or by Z2D3/3E5.
- the antibody may comprise the amino acid sequences of a human framework region and of a constant region from a human antibody.
- Such chimeric antibody may be a genetically engineered hybrid neomolecule conjugated to the enzyme or to the proenzyme, such that the neomolecule is partially an antibody and partially an enzyme.
- the chimeric antibody may also be a bifunctional antibody.
- the bifunctional antibody is usually produced by a quadroma.
- the quadroma is derived from the fusion of a hybridoma cell line Z2D3 or Z2D3/3E5 and a hybridoma secreting a monoclonal antibody binding an enzyme.
- the enzyme may be any enzyme capable of digesting a component of the plaque.
- the enzyme is a proteinase, an elastase, a collagenase, or a saccharidase.
- the enzyme is fibroblastic collagenase, gelatinase, polymorphonuclear collagenese, granolocytic collagenase, stromelysin I, stromelysin II, or elastase.
- the subject invention also provides a composition comprising an amount of the above-described reagent effective to digest a component of atherosclerotic plaque and a physiologically acceptable carrier.
- the subject invention provides a method for reducing the amount of atherosclerotic plague in a blood vessel which comprises contacting the atherosclerotic plaque with the reagent for digesting atherosclerotic plaque described above, under conditions and in an amount such that the reagent binds to and digests, a component of plaque.
- the subject invention also provides a method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises: a) contacting normal lumen with an antibody which specifically binds to normal intima and/or media and has bound thereto an inhibitor of an enzyme under conditions such that the antibody binds to normal intima and/or media; and b) contacting the atherosclerotic plaque with the reagent for digesting atherosclerotic plaque under conditions such that the reagent binds to the atherosclerotic plaque.
- the antibody which specifically binds to normal intima and/or media is provided in a reagent for use in protecting normal arterial tissue from an enzyme capable of digestion of atherosclerotic plaque.
- Such reagent which is bound to an inhibitor of an enzyme capable of digesting atherosclerotic plaque, comprises an antibody which binds an antigen synthesized or present in such normal tissue, such as the monoclonal antibody produced by hybridoma Q10E7, as well as a recombinant polypeptide which comprises an amino acid sequence which is substantially the same as the amino acid sequence of the hypervariable region of the monoclonal antibody produced by hybridoma Q10E7, or a chimeric or humanized antibody or fragment thereof comprising the recombinant polypeptide.
- This invention further provides a method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises: a) contacting the atherosclerotic plaque with a reagent under conditions such that the reagent binds to the plaque so as to form a reagent-plaque complex, which reagent is capable of specifically binding to both the plaque and to a proenzyme which, when cleaved, is converted into an enzyme whose substrate is a connective tissue present in atherosclerotic plaque, and which enzyme is capable of dissolving a component of the plaque; b) contacting the reagent-plaque complex with the proenzyme to which the reagent specifically binds under conditions such that the proenzyme is bound to the reagent so as to form a proenzyme-reagent-plaque complex; and c) contacting the proenzyme-reagent-plaque complex with an agent which is capable of specifically cleaving the proenzyme so that the proenzyme is converted into the
- the subject invention further provides a method for reducing the amount of atherosclerotic plaque in a blood vessel which comprises a) contacting the atherosclerotic plaque with a reagent such as the reagent described above for digesting atherosclerotic plaque under conditions such that the reagent binds to the plaque so as to form a reagent-plaque complex, which reagent is bound to both the plaque and to a proenzyme which, when cleaved, is converted into an enzyme whose substrate is a connective tissue present in atherosclerotic plaque, and which enzyme is capable of dissolving a component of the plaque; and b) contacting the proenzyme-reagent-plaque complex with an agent which is capable of specifically cleaving the proenzyme so that the proenzyme is converted into the enzyme under conditions such that the enzyme digests the plaque.
- a reagent such as the reagent described above for digesting atherosclerotic plaque under conditions such that the reagent binds
- the reagent is a bifunctional antibody.
- the bifunctional antibody may be produced by any method known in the art including chemical linkage of fragments, and recombinant genetic engineering.
- the bifunctional antibody is produced by a quadroma, wherein the quadroma is derived from the fusion of a hybridoma cell line comprising the monoclonal antibody produced by hybridoma Z2D3 or Z2D3/3E5 or related cell line and a hybridoma secreting a monoclonal antibody binding an enzyme.
- the proenzyme be a proenzyme of granulocytic collagenase, fibroblastic collagenase, or stromelysin.
- the agent of step (c) is plasmin.
- the plasmin may be obtained by treating the subject with tissue plasminogen activator under such conditions so as to cleave plasminogen into plasmin.
- the subject invention provides a reagent for use in ablating atherosclerotic plaque which comprises an antibody which specifically binds to atherosclerotic plaque bound to a chromophore capable of absorbing radiation having a plaque ablating wavelength.
- the antibody is a monoclonal antibody such as that produced by hybridoma 15H5 or 17H3and more preferably, the monoclonal antibody is produced by hybridoma Z2D3 or hybridoma Z2D3/3E5 or related daughter cell line.
- the chromophore absorbs light having a wavelength of from about 190 nm to about 1100 nm. Such chromophores are well known in the art. Accordingly, the choice of chromophore is readily determinable to one skilled in the art although a preferred embodiment is wherein the chromophore is fluorescein, rhodeunine, tetracycline, or hematoporphyrin.
- the subject invention further provides a composition comprising an amount of the above-described reagent effective for use in ablating atherosclerotic plaque and a physiologically acceptable carrier.
- This invention provides a method for ablating atherosclerotic plaque which comprises: a) contacting atherosclerotic plaque with an effective amount of the reagent for use in ablating atherosclerotic plaque described hereinabove so that the reagent binds to the atherosclerotic plaque forming an atherosclerotic plaque-reagent complex; b) exposing the resulting complex to radiation having a plaque ablating wavelength under conditions such that the light is absorbed by the chromophore at a sufficient energy to ablate the atherosclerotic plaque.
- the subject invention further provides a method for ablating atherosclerotic plaque present in a blood vessel which comprises: a) contacting the normal lumen with an antibody which specifically binds to normal intima and/or media and has bound thereto a moiety capable of reflecting radiation of the plaque ablating wavelength; b) contacting the atherosclerotic plaque with the reagent for use in ablating atherosclerotic plaque described hereinabove under conditions such that the reagent binds to the atherosclerotic plaque; and c) exposing the atherosclerotic plaque to radiation having plaque ablating wavelength, thereby ablating the plaque.
- the antibody which specifically binds to normal intima and/or media is a monoclonal antibody produced by hybridoma Q10E7 (ATCC Accession No. HB 10188).
- the subject invention also provides a reagent for use in treating atherosclerosis which comprises an antibody which binds specifically to atherosclerotic plaque bound to drug useful in treating atherosclerosis.
- the antibody is the monoclonal antibody produced by hybridoma Z2D3 (ATCC Accession No. HB9840).
- the subject invention provides a method of treating atherosclerosis in a subject which comprises a) administering to the subject an antibody which specifically binds to normal intima and/or media and which has bound thereto an inhibitor of a drug useful in treating atherosclerosis; and b) administering to the subject an amount of the reagent described above effective to treat atherosclerosis.
- the antibody for use in protecting normal arterial tissue from a drug useful in treating atherosclerosis is a monoclonal antibody produced by hybridoma Q10E7 (ATCC Accession No. HB 10188) which has bound thereto an inhibitor of a drug useful in treating atherosclerosis.
- the subject invention provides a method of treating atherosclerosis which comprises blocking the synthesis of an atherosclerotic plaque specific antigen.
- the blocking of the atherosclerotic plaque antigen may be accomplished in several ways.
- One embodiment of this method is wherein the synthesis of the antigen is blocked by using an antisense nucleic acid which specifically binds to a nucleic acid encoding the antigen, the expression of which is associated with synthesis of the antigen.
- the synthesis of the antigen is blocked by inhibiting an enzyme involved in the synthesis of the antigen.
- the subject invention also provides a method of treating atherosclerosis which comprises blocking the binding of an antibody, such as an auto-antibody to the atherosclerotic antibody plaque antigen.
- This method may encompass any of the methods known to one skilled in the art.
- One embodiment of this method comprises blocking the binding of the auto-antibody to the antigen by contacting the antigen with an excess of antibody.
- Another embodiment of this method comprises blocking the binding of the auto-antibody to the antigen by contacting the auto-antibody with an excess of an anti-idiotype antibody made thereto.
- the subject invention further provides a method for diagnostic analysis comprising the steps of: a) obtaining a value for the body mass index (BMI) of a patient; b) obtaining a value for the concentration of an antigen or other serum or plasma analytes associated with a pathological condition or an antibody which binds with the antigen; c) plotting the body mass index of the patient against the antigen or antibody concentration of the same patient; and d) comparing the resulting value against a set of reference values to determine whether the resulting value exceeds the reference value and thereby indicates the presence of a pathological condition.
- BMI body mass index
- This method is a generic method may be used instead of conventional methods which just reveal positive or negative results in testing whether a patient has a predisposition toward such pathological conditions as cancer and atherosclerosis.
- the method is preferable used wherein the antigen is an antigen synthesized by, or present in, atherosclerotic plaque ( Figure 24), or wherein the antibody is an antibody which specifically binds to such an antigen ( Figure 23).
- the body mass index (BMI) is obtained by dividing a subject's weight by their height 2 .
- the following method is a one-step procedure for antigen affinity purification by means of 15H5 Ab gel:
- the plaque-matrix-specific antigen (Z2D3 Ag) was discovered when its identifying monoclonal antibody, Z2D3, was screened positive by immunohistology on atherosclerotic human coronary arteries.
- the Z2D3 antibody stained the plaque matrix without staining normal artery.
- the immunogen used in the hybridoma program that produced this antibody was affinity-purified material obtained from homogenized human plague using the monoclonal antibody 15H5.
- the 15H5 antibody recognizes the carbohydrate autoantigen produced by normal and plaque-derived smooth muscle cells (Lamaziere, J.M., et al. Atherosclerosis (Ireland) 74 (1-2): 115 (1988)).
- the Z2D3 antibody was further screened on a variety of human tissues using 5 ⁇ unfixed frozen tissue sections (Calenoff, E., et al., unpublished results).
- the normal tissue staining was confined to the cytosol without extracellular manifestations.
- the vast portion of staining within atherosclerotic plaque was extracellular, diffusely manifest throughout the connective tissue matrix in addition to staining the cytosol of the plaque smooth muscle cells.
- the plaque was then cut into 2 ⁇ 4mm fragments and freeze-dried into small flakes (to remove as much free water as possible).
- the flakes were embedded in O.C.T. medium (Miles Labs, Elkhart, IN), blocked, and snap frozen.
- the frozen tissue block was mounted in a tissue section cryostat and 5 ⁇ tissue sections were cut.
- the tissue sections were mixed with 10 parts acetonitrile in a glass beaker.
- the glass beaker was tightly covered and the contents stirred vigorously for 18- 24 hours at room temperature.
- the stirred contents were then centrifuged at 20,000 g, 4°C for 1 hour and the precipitate discarded.
- the acetonitrile solution was filtered through a 0.22 ⁇ nylon filter (Millipore, Bedford, MA) yielding pale, yellow colored supernatant.
- Ion Exchange Chromatography Ion-exchange chromatography was performed on a Mono Q HPLC column (Pharmacia) as per the method of Mansson (J.E. Mansson, B. Rosengren, L.
- Example was applied in multiple 0.5 uL aliquots per lane, and dried each time with a hair dryer.
- the loaded plate was then placed in a container containing the mobile phase (chloroform, methanol, glacial acetic acid and water in the volume ratios 25:15:4:2)
- the mobile phase was approximately 3mm from the top of the plate, the plate was removed and allowed to air dry at ambient temperature.
- the plates from various TLC runs were stained individually with iodine staining or immunoperoxidase staining employing the Z2D3 antibody. The iodine staining was done by placing iodine crystals in a covered glass container and then incubated for 10-30 min.
- the dry TLC plate was placed in the container. When the iodine reactive spots reached the desired degree of darkness, the plate was removed and inspected. A single spot at the upper edge of the mobile phase was observed. It was confirmed to be the Z2D3 antigen by running duplicate plates through an immunoperoxidase staining procedure employing the Z2D3 antibody and a negative control antibody.
- the washed plates were then incubated in a 1:500 dilution of goat anti-mouse IgM/horse radish peroxidase conjugate (Tago, Burlingeune, CA.), for 3 hours with gentle agitation.
- the plates were then washed in casein buffer followed by 3 washes in TBS.
- the washed plates were then immersed in the substrate solution which consisted of 8ml of 3 mg/mL 4-chloro-naphthol in methanol, 32 mL of TBS and 20 uL 30% H2O2.
- the plates were developed for 10-20 minutes and the reaction stopped by rinsing the plates with deionized water. A developed spot was seen at the mobile phase line on the Z2D3 plate but none on the nonspecific antibody plate.
- the Z2D3 antigen appears to be a small, lipid containing molecule. It is probably not a sphingolipid because of its resistance to the usual acid hydrolysis conditions but is perhaps a neutral lipid or a proteolipid.
- Various antigen fractions in either ethanol or other organic solvents were applied in 100 uL samples into the wells of Immulon 4 microtiter plates (Dynatech, Chantilly, Va.) and the organic solvents evaporated by blowing gently with bottled nitrogen gas or air. The dried plates were then blocked by applying 200 uL of the casein/TBS solution (wash buffer) for 1 hour.
- the plates were then washed 4 ⁇ and 100 uL aliquots of 5 ug/mL Z2D3 antibody diluted in wash buffer applied to each test well.
- the plates were covered and incubated at 37° for 1 hour. They were washed 4x and 100uL of goat anti-mouse IgM/peroxidase conjugate, diluted 1:100 applied.
- the plates were incubated for 1 hour at 37° and then washed.
- 100 ul of TMB substrate (Kirkegaard and Perry, Gaithersburg, MD.) was applied to each well and incubated for 1 hour at room temperature. 50 uL of 1M HCl was applied to each well to stop the substrate catalysis and the plates read at 450 nm on a Molecular Devices ELISA reader (Molecular Devices, Menlo Park, CA) .
- This molecular marker of atherosclerotic plaque makes possible the development of novel diagnostic and therapeutic reagents for improving the clinical care of patients afflicted with coronary and/or cerebrovascular atherosclerosis.
- imaging agents comprising Fab fragments labeled with radionuclides such as technetium to be used in nuclear imaging or those labeled with paramagnetic molecules to be used in MRI imaging.
- Targetable therapeutic reagents such as neomolecules could be constructed which possess antibodylike targeting and enzymatic activity which would yield a controlled catalysis and reduction of the connective tissue content in atherosclerotic plaque, thereby relieving focal arterial obstruction and preventing myocardial infarction and/or stroke (Kates, M., Techniques of Lipidology (Elsevier, New York, ed. 2, 1986)).
- Method 2
- CsCl fraction number one Z2D3
- fraction number two Q10E7
- Either CsCl fraction number one (Z2D3) or fraction number two (Q10E7) is/are dialyzed in 3500 MW tubing against 20 mM Tris-Hcl/7M Urea, pH 7.5, to achieve a 50,000x dialysis effect.
- Semi-prep column 10-150 mg protein load range 4.
- the eluting buffers are 20mM Tris-HCl/7M Urea, pH 7.5 (A), and 20mM Tris-HCl/ 7M Urea/IM NaCI, pH 7.5 (B).
- each sample from each tube is diluted in 20 mM Tris-HCl/3.5M Urea, pH 7.5, and used to coat wells on microtiter plate corresponding to each tube collected. Plates are incubated overnight at RT.
- the antigen-containing mixture is added to agarose gel, coupled to Z2D3 or Q10E7 monoclonal antibody (depending on which antigen requires purification).
- This agarose gel/antigen mixture is gently mixed in a shaker at 4oC overnight.
- the gel is washed with 20 gel volumes of 20mM Tris-HCl/0.15M NaCI, pH 7.4(Tris). 5.
- the antigen is eluted with 0.1M glycine HCl buffer, pH 2.5.
- the eluted antigen is dialyzed against Tris buffer.
- the affinity purified antigen is concentrated using an Amicon concentrator with 10,000 MW filter.
- the concentrated antigen is filtered using 0.45 ⁇ filter and loaded onto a Bio-Sil TSK-400 column (Z2D3 antigen) or Bio-Sil TSK-250 column (Q10E7 antigen) equilibrated with 0.1M potassium phosphate buffer, pH 7.0 (Bio-Sil columns are sold by Bio-Rad).
- the major macromolecules of Z2D3 antigen are eluted at greater than 200,000 MW from the Bio-Sil TSK-400 column.
- the Q10E7 antigen is eluted from the Bio-Sil TSK-250 column at greater than 150,000 MW.
- Affinity purified atherosclerotic plaque antigen was coated onto polystyrene microtiter plates (Immunlon II). Sample was diluted in 100 mM NaPO 4 /400 mM NaCl/pH 6.9 and 100 ⁇ l was applied to each well and then incubated overnight at 4oC. Plates were blocked and then washed with PBS containing 0.1% Triton X-100 and 0.05% Tween-20 (for lectin study) or casein buffer (for commercial antibody study). Biotinylated lectins (from Vector or Sigma) were diluted to a final concentration of 1-10 ⁇ g/ml and 100 ⁇ l applied to wells coated with athero-antigen for 2 hrs at 37°C.
- Bound lectins were detected using an Avidin-Peroxidase conjugate [ABC from Vector Labs].
- Commercial polycional and monoclonal antibodies were diluted with casein buffer (1/100 to 1/2000) and incubated with coated antigen (prepared as above) for 2 hrs. at 37°C.
- Appropriate peroxidase conjugated second antibodies were then applied to detect binding of commercial antibodies to the coated athero-antigen.
- TCA trichoroacetic acid
- One volume of partially purified atherosclerotic plaqueantigen was mixed vigorously with one volume of chloroform, and centrifuged 5 minutes at 2000 RPM. The upper aqueous layer was removed and extracted again with chloroform. After a second centrifugation, the aqueous layer was assayed by ELISA.
- Affinity purified atherosclerotic plaque antigen from plaque or serum was mixed with a wide array of hydrolytic enzymes, the specific reaction buffers were those suggested by the manufacturer. All reactions were done in a total volume of 1.0 - 1.5 ml, incubated overnight at 37°, then boiled for 5 minutes to stop the reaction. Samples were filtered (0.45 ⁇ ) and injected onto an HPLC column (TSK-400600 mm ⁇ 7.5 mm Bio-Rad) for molecular sieve fractionation in 0.1 M KPO 4 , pH 7.0.
- Atherosclerotic plaque antigen was exposed to the following list of denaturants and then returned to its original buffer [PBS] by dialysis.
- Partially purified atherosclerotic plaque antigen (0.5 ml in 0.1 M KPO 4 , pH 7.0) was mixed with 0.44 guanidine-HCl (7M final concentration) and 250 ⁇ g of dithiothreitol. The pH was adjusted to 8.6 and the sample left for 1 hr. at R.T. Then 16 mg of iodoacetamide was slowly added and pH maintained at 8.5 with NaOH as needed. After 1 hr. at R.T. this seunple was run on a TSK-400 HPLC gel filtration column (as described above) and individual fractions were tested for atherosclerotic plaque antigen by ELISA and compared to fractions collected from unreacted antigen.
- Antigen and control samples were prepared by affinity chromatography. Affinity resins were prepared by coupling Vasocor monoclonal antibody 15H5 to Bio-Rad Affigel 10 using published methods. Plasma samples were incubated with the resin in a batchwise procedure. After washing, the bound antigen was eluted with 0.1 M glycine buffer pH 2.5. The antigen solution was then neutralized and dialyzed against PBS. Sample Preparation:
- Salts were removed by extensive dialysis against purified water at 4°C. Each sample was concentrated by lyophilization and the residue redissolved in a minimal volume of purified water.
- Ultra high purity monosaccharide standards were obtained from Pfansteiehl Laboratories Inc., Waukegan, IL. Standard solutions and dilutions thereof were all prepared in purified water. Aliquots of each standard were stored at -80oC until use.
- the Dionex instrument was expressly configured for monosaccharide analysis, consisting of a reagent delivery module, micro-injection valve, pulsed eunperometric detector with gold electrode, and CarboPac PA-1 analytical column. Data were collected on a Dionex 4270 Integrator.
- the column was thoroughly equilibrated in 15 mM NaOH in purified water. Hydrolysate residue were redissolved in purified water just prior to injection. The bound monosaccharides were eluted from the CarboPac column with a linear gradient of NaOH in purified water.
- Figure 20 illustrates a chromatographic blank run with just distilled water.
- Figure 21 illustrates a chromatographic run with seven standard monosaccharides.
- Figure 22 illustrates a chromatographic blank run with the auto-antigen affinity purification with the 15H5 monoclonal antibody.
- the gel is ten washed with the Coupling Buffer, 0.1 M Acetate Buffer, pH 4.0, containing 0.5 M NaCl, and washed twice with Coupling Buffer.
- the protein-Sepharose conjugate is now ready for use and can be stored at 4 to 8°C. Cyanogen bromide can be added to the buffer solution as a bacteriostat.
- IgG Antibody Adsorption from Plaque Supernatant A column is packed with 25 ml of Sepharose gel conjugated to anti-IgG antibody prepared in accordance with the above procedure containing a total of about 129 mg of anti-IgG antibody.
- the column is equilibrated with from 2 to 3 volumes of buffer (0.15 M PBS, pH 7.2), and the sample is then applied to the column.
- the flow rate of eluting buffer (0.15 M PBS, pH 7.2) is 125 ml/hr.
- the eluted fractions containing antibody are collected until peak activity disappears.
- the column is then washed with sodium acetate buffer solution, pH 4.0 (Eluting Buffer) to desorb immunoaffinity bound IgG antibody.
- the column is eluted a rate of 15-20 ml/hr, collecting the eluted samples and retaining peak fractions.
- the peak fractions are dialyzed against 0.15 M PBS, pH 7.2, for 24-36 hr at 4°C with multiple buffer changes.
- IgE Antibody Adsorption from Plague Supernatant The above procedure is repeated with a column packed with 7.5 ml of Sepharose gel conjugated to anti-IgE antibody prepared as stated above. The flow rate of Eluting Buffer is 15-20 ml/hr. IgA Antibody Adsorption from Plague Supernatant
- Polycional Anti-Plaque Antibodies Polycional antiserum against atheroslerotic plaque antigen is elicited in rabbits using the immunization techniques and schedules described in the literature, e.g. [Stollar, Methods of Enzymology, 70:70 (1980)]. The antiserum is then screened in a solid phase assay similar to that used for monoclonal antibodies, e.g. [Lange et al., Clin. Exp. Immunol., 25:191 (1976) and Pisetsky et al., J. Immun. Methods. 41:187 (1981).] The initial screening criterion would be binding to atherosclerotic plaque antigen.
- Polycional anti-plaque antibody must be prepared as follows: Rabbits may be injected intramuscularly with a mixture of 0.5 mg of plaque antigen prepared by the procedure described hereinabove in 0.2 ml of 0.15 M sodium chloride solution and 0.8 ml of complete Freund's adjuvant. The immunization is repeated for 14 days and then each week for 3 weeks. After a further 10 days have passed, blood is removed from the rabbits, and antiserum is recovered from the blood by allowing it to coagulate and removing the clot. Repeating the above procedure but replacing the antibody reagent with the plaque antigen yields horseradish peroxidase or alkaline phosphatase labeled plaque antigen.
- the IgG fraction of the antisera is purified further by affinity chromatography on a column containing a resin on which the anti-plaque antigen is immobilized.
- mice monoclonal antibodies to the plaque antigen are obtained using standard procedures of Galfre and Milstein, [Methods in Enzym., 73:1 (1981)].
- the monoclonal antibodies are screened using a modification of the techniques described in the literature, e.g., [Lange et al., Clin. Exp. Immune, 25:191 (1976)] and Pisetsky et al. [J. Immun. Methods., 41:187 (1981)].
- a monoclonal antibody should bind to the plaque antigen with high affinity (preferably, K A 10 10 M -1 ).
- Mouse monoclonal antibody is purified in a two step procedure.
- the neat ascites fluid is applied to a column of Affi-Gel Blue resin (Bio-Rad Laboratories, Richmond, CA) equilibrated with 10 mM Tris-HCl, 0.15 M NaCl, pH 8.0, and eluted with the buffer. This step removes albumin, which is retained on the column.
- the final step in the purification is application to a DEAE-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) and elution with a linear gradient of 10 mM Tris-HCl, pH 8.0, to 10 mM Tris-HCl, 100 mM NaCl. This gives purified mouse monoclonal antibody free from contaminating serum proteins such as albumin and transferrin.
- the class switch was from an IgM isotype cell line Z2D3 (ATCC Accession No. 9840) with specificity for atherosclerosis plaque antigen, to an IgG isotype cell line (Z2D3/5C5) with the same specificity.
- Z2D3/5C5 is an example of several daughter cell lines of Z2D3. Such daughter cell lines also include Z2D3/3E5 (ATCC Accession No. HB 10485).
- the IgM isotype Z2D3 hybridoma cell line was prepared by fusing Balb/c splenocytes with the SP2 Myeloma cell line. (See Journal of Immunology, Vol. 131 No.
- Z2D3 was screened initially for IgG producing cells. 100 cells were plated/well in 96 well Falcon plates for a total of 10 plates. At day 8 supernatants were collected and tested for IgG. 96 wells were coated overnight at 4oC with 50ng/well of goat anti-mouse IgG. ( ⁇ chain specific) reagent Zymed 62-6600). Wells were washed X 4 with PBS with 0.05% Tween (wash buffer), and 50 ⁇ l of supernatant from the plated cells was added.
- the sensitivity of the assay enabled one positive cell in 100 to be detected easily. Initially 3 positive wells were detected. The well (8G2) producing the highest signal was further enriched by subcloning as follows: This positive well was then resuspended in 100 ml of medium containing 9% Fetal Calf Serum, and plated in 5, 96-well plates at 200 ⁇ l/well. Supernatants from these wells were tested as above 8 days later, and 70% of the wells were positive for IgG. The well (1A12) with the highest signal for IgG was chosen for additional subcloning. Cells in the well were suspended by pipetting and 20 ⁇ l of the suspension was diluted into 100 ml of medium with 9% Fetal Calf Serum. The suspension was plated 200 ⁇ l/well in 5 plates, with approximately 3 cells/well.
- the IgG class was confirmed and the subclass determined using a Sublsotyping Kit (Hyclone EO5051-K).
- Z2D3/5C5 is IgGl.
- TMB substrate (using equal volumes mix TMB substrate with peroidase solution B). 10. Add 100 ⁇ l substrate into each well and react at R.T. for 60 minutes.
- optical density (O.D.) number is directly proportional to the concentration of antibody in tested sample.
- TMB substrate (using equal volumes mix TMB substrate with peroxidase solution B).
- optical density (O.D.) number is directly proportional to the concentration of antigen in the tested sample.
- Inhibition Assay Protocol 1 Apply 100 ⁇ l different concentration of HCAD or in-house monoclonal antibody (17H3 Ab., Z2D3 Ab., 15H5 Ab., and normal mouse IgM as a mono Ab. control into antigen-coated wells in order to pre-block wells. At the same time, add 100 ⁇ l 10 mM PBS buffer into antigen-coated wells as a noninhibition control). The plate is covered with parafilm. 2. Incubate plate at R.T. for two hrs., and then at 4oC overnight.
- Inhibition % - 100% - Assay well mean O.D. ⁇ 100% PBS control well Mean O.D. Immunoassay Procedure for Atherosclerotic Plague Antigens
- the microtiter plates are then read in either a colorimetric reader (Molecular Devices) or a fluorometer (3M Diagnostics) every 10 minutes until the the maximum reading or 1 hour is reached.
- microtiter plate coated with atherosclerotic plaque antigen 100 microliters/well of human serum is applied (Sample may be diluted). The plates are covered to prevent drying and incubated for 2 hours, and the residual solution is removed, and the plates washed three times with casein wash buffer. 100 Microliters/well of a solution of affinity purified goat anti-IgG, IgM, IgA, or IgE conjugated to either horseradish peroxidase or alkaline phosphatase (appropriately diluted) is applied to each well. The plates are covered to prevent drying and incubated for 2 hrs. The anti-IgG, IgM, IgA, or IgE solution is removed and the plates washed three times with casein wash buffer.
- the microtiter plates are then read in a colorimetric reader (Molecular Devises) or a fluorometer (3M Diagnostics) every 10 minutes until the first maximum reading or 1 hr is reached.
- Microliters of prepared dilutions of plaque antigen are applied to the surface of IMMULON II microtiter plates (Dynatech).
- the coating solution dilutions are 1:10, 1:100, 1:1000 and 1:10,000.
- the plates are tapped gently and to insure the coating solution covers the bottom of each well completely.
- the well are incubated at 4oC overnight in a covered, humidified box.
- the coating solution is discarded and 200 microliters PBS is added per well.
- the wells are then incubated at room temperature for 1 hr in a humidity box, then washed with 200 microliters of Wash Buffer (PBS, 0.5% Tween and 0.02% sodium azide), and stored in a humidity box at 4°C until use.
- Wash Buffer PBS, 0.5% Tween and 0.02% sodium azide
- Antibody Coated Microtiter Plate 100 Microliters of prepared dilutions of anti-plaque antibody are applied to the surface of IMMULON II microtiter plates (Dynatech).
- the coating solution concentrations are selected to be from 1-5 micrograms/well but can be varied up or down depending upon the selection of other reagents and immunoassay procedures to be followed.
- the plates are tapped gently and to insure the coating solutin covers the bottom of each well completely.
- the wells are incubated at 4°C overnight in a covered, humidified box.
- the coating solution is discarded, and 200 microliters of 1% BSA in PBS is added per well.
- the wells are then incubated at rm temp for 1 hr in a humidity box, and the BSA solution is removed.
- the wells are the washed with 4 times with 200 microliters of Wash Buffer (PBS, 9.5% TWEEN, and 0.02% sodium azide), and stored in a humidity box at 4°C until use.
- Wash Buffer PBS, 9.5% TWEEN, and 0.02% sodium azide
- the following method is an one-step procedure for coupling peroxidase to a monoclonal antibody (e.g. the monoclonal antibody produced by hybridoma 15H5 or 17H3):
- Conjugate is filtered by 0.2 ⁇ filter.
- the antibody can be labeled with 1-131 by the Pierce lodobead method described by Rosebrough, S. (supra, p 575). 100 ⁇ l (microliters) of 0.2 M PBS, pH 7.0, is added to 150 ⁇ g of antibody, followed by the addition is incubated for 10 min. The solution is removed with a pipette and reserved, and the beads are washed with 100 ul of 0.2 M PBS, pH 7.0. The solution and wash buffer from the beads are combined. To separate the free iodine, the solution is washed exhaustively with a CENTRICON C-30 filter. Approximately 60% of the original I-131 is bound to the antibody.
- DTPA is coupled to antibody by the method of Hnatowich, D. et al. [Journal of Immunolical Methods, 65:147 (1983)].
- the bicyclic anhydride of DTPA is prepared as described by Hnatowich D. et al., [Int. J. Appl. Radiat. Isot. 33:327 (1982)] and is stored as the solid in a desiccator at R.T.
- a suspension of the anhydride in dry chloroform or ether (0.01 mg/ml) is prepared and an aliquot evaporated under nitrogen in a clean, dry teat tube, from 10-20 ⁇ l of the antibody solution in 0.05 M bicarbonate buffer in saline, pH 7.0 - 7.5, is immediately added and the contents agitated for 30 to 60 sec.
- the coupled antibody is to be purified before labeling, the preparation is diluted to about 0.2 ml with the above buffer and purified on a 5 cm gel filtration column (G-50; Roche Diagnostics, Nutley, NJ) using saline eluant. The purification takes about 5 min and provides a product which is approximately 95% pure.
- Antibody is dialyzed overnight against pH 9.5 carbonate/bicarbonate buffer solution. The concentration is determined (for example by otical density at 280nm. A solution of luorescein isocyanate (1.0/mg/ml) in DMSO is prepared, and the desired volume (1-10% of total protein solution volume) is added to the antibody solution dropwise, with stirring. The reaction proceeds for two hours, shielded from light. The product is purified by gel filtration on SEPHADEX G-25 gel in PBS containing 0.1% NaN3 to separate the unreacted or hydrolyzed fluorochrome. The absorbance of the conjugate is measured at 280 nm and at 495 nm to yield a solution of fluorescein labeled antibody.
- Antibody is dialyzed overnight against pH 9.5 carbonate/bicarbonate buffer solution as described in Example 7.
- a solution of rhodamine isocyanate (10.0 mg/ml) in DMSO is prepared, and the desired volume (1-10% of total protein solution volume) is added to the protein solution dropwise, with stirring. The reaction proceeds for two hours, shielded from light.
- the product is purified by gel filtration on SEPHADEX G-25 gel in PBS containing 0.1% NaN3 to separate the unreacted or hydrolyzed fluoroschrome. The absorbance of the conjugate is measured at 280 nm and 550 nm to yield rhodamine labeled antibody.
- Antibody which binds specifically to atherosclerotic plaque (1 m mole) is dialyzed st 4°C against a buffer solution of 0.01 M PBS, pH 6.8 overnight. To this solution is added 50 nmole of 3-carboxy-7-hydroxycoumarin. The solution is added 50 mnole of 3-ceurboxy-7-hydroxycoumeu:in. The solution is cooled in an ice bath and added with 50 nmole of 1-ethy1-3-(3-dimethylaminopropy1)carbodiimide hydrochloride. After addition the mixture was stirred at 4oC for one hour and chromatographed on a 2.5 c 50 cm column of SEPHAEX G-50. The absorbance of the conjugate is monitored at 345 nm to yield a solution of coumarin labeled antibody. Nile Blue A Labeled Antibody
- Nile Blue A (350) mg) is diazotized according to the procedure described above.
- the solution containing the diazoni ⁇ m salt of Nile blue A is added dropwise to anti-plaque antibody (0.05 m mole) in on 0.1 M PBS, pH 8.0. After addition the mixture is purified on a 2.5 ⁇ 50 cm SEPHADEX column. The absorbance of the eluate is monitored at 628 nm to yield the Nile blue A labeled antibody.
- Antibody 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (50 m mole) is added to a mixture of hematoporphyrin (50 m mole) and anti-plaque antibody (1 m mole) in 0.01 M PBS, pH 6.8. After addition, the mixture is stirred at 4oC for 2 hours. The mixture is then purified with a SEPHADEX G-50 column to yield the hematoporphyrin conjugated antibody.
- the NHS ester of carboxymethyltetracycline is prepared as described above.
- Enzyme Labeled Anti-Plaque Antibody may be conjugated with alkaline phosphatase following the modified procedure of O'Sullivan, M. et al. [Analytical Biochem., 100:100 (1979)].
- Horseradish peroxidase is conjugated to anti-plaque antibody in accordance withe procedure of Nygtren, H. et al. [Medical
- HRP horseradish peroxidase
- the GA-HRP complex is, in a second step, mixed with the antibody in 0.05 M carbonate: bicarbonate buffer, pH 9.5, containingg 0.15 M NaCI, at different IgG:HRP ratios for 16-64 hr at 4°C. The reaction is stopped by the addition of lysine to a final concentration of 0.02
- Trypsin-labeled Antibody m-Maleimidobenzoyl N-hydroxysuccinimide in dry dimethylformamide (100 ul, m mole/1) is added to the purified antiplaque antibody. The resulting mixture is stirred at room temperature for 30 min.
- the antibody solution is fractionated on a SEPHADEX G-50 column with PBS as eluant. To the pooled antibody solution is added trypsin (13 mg) at room temperature. After addition, the mixture is stirred for another 2 hr. 2-Mercaptoethanol is a added to a final concentration of 2 mM/1, and the solution stirred for a further 30 min.
- the conjugate is dialyzed overnight against PBS (3 ⁇ 2 liters) to yield the trypsin-labeled antibody.
- Papain Labeled Antibody Equimoleu amount of purified the anti-plaque antibody in papain are mixed in a solution of 0.1 M sodium-potassium. To this solution is added 1 vol.% glutaraldehyde solution in phosphate buffer. After addition, the mixture is stirred at room temperature for 3 hours and finally dialyzed overnight against 0.1 M. PBS, pH 8.0, at 4°C. The mixture is further purified on a SEPHADEX G-50 column to yield the papain-labeled antibody.
- hyaluronidase 5 mg
- phenyl isothicocyanate to protect the free amino groups on the hyaluronidase molecule.
- the solution is stirred gently at room temperature for 1 hour.
- a solution of sodium periodate (0.06 M in distilled water) is added and the mixture stirred gently for 30 minutes.
- One ml of 0.16 M of ethylene glycol in distilled water is added, and the solution stirred for another 1 hour at room temperature.
- dialyzing against 0.01 M of sodium carbonate buffer, pH 9.5, at 4oC (3 ⁇ 1 liter) the mixture is mixed with purified anti-plaque antibody.
- the reaction mixture is stirred for 2-3 1 hours and treated with 5 mg of sodium borohydride. The mixture is allowed to stand at 4°C overnight. Following dialysis against PBS buffer, the dialyaia against PBS buffer, the mixture is chromatographically purified with a 1.5 ⁇ 85 cm (BN) SEPHADEX G-100 column to yield the hyaluronidase antibody.
- BN 1.5 ⁇ 85 cm
- Collagenase I (65 lysines) collagenase II (50 lysines) is dissolved in 0.05 M phosphate buffer, pH 8.0. To this solution is added m-maleimidobenzoly N-hydroxysuccinimide in anhydrous dimethylformamide (100 ul, 8 m mole/liter). After addition, the mixture is stirred for 30 minutes.
- the enzyme solution is first fractionated SEPHADEX G-50 column with PBS as eluant and teated with purified anti-plaque antibody. The mixture is stirred for 2 hours at room temperature and added with 2-mercaptoethanol to a final concentration of 2 m mole/1. The mixture is stirred for a further 30 minutes and chromatographically purified on a SEPHADEX G-50 column to yield collagenase conjugated anti-plaque antigen antibody.
- Beta, anti-collagenase and anti-(normal vascular epithelium) antibody are coupled with glutaraldehyde by the procedure described hereinabove.
- the final reaction mixture is purified with a 2.5 ⁇ 50 cm SEPHADEX G-200 column to yield the beta 1 anticollagenase labeled anti-(normal vascular epithelium) antibody.
- the descending aorta segment from the thorax region was used as normal control relative to the de-endothelialized abdominal segment of descending aorta.
- the aortic segments were cleaned of blood and dissected enfaced.
- Monoclonal antibody Z2D3-5C5 F(ab') 2 fragments specific for an atheroma connective tissue antigen were used for non-invasive imaging of atheromatous lesions in an experimental rabbit model produced by balloon catheter deendothelialization of the descending aorta followed by a high cholesterol and fat diet. Seven weeks later, 3 animals were injected intravenously with In-111 Z2D3. 1-125 Z2D3 and 1-125 nonspecific monoclonal F(ab') 2 were also injected in each one of these animals. Images were recorded at 15 min, 24 H, and 48 H. The normal (N) and lesioned (L) segments were weighed, counted by gamma scintigraphy and expressed as mean percent injected dose per gram.
- Unfixed frozin tissue section (5-6 ⁇ m thick) preferably freshly cut and stored overnight at -80oC.
- Bovine Serum Albumin BSA (Sigma #A-7030).
- Biotinylated horse antimouse IgG (Vector - Peroxidose Mouse IgG PK 4002).
- Methyl alcohol absolute low acetone (Mallinckrodt #3016-4 or equivalent).
- Trizma base (Sigma #T-1503 or equivalent).
- PBS Phosphate-buffered saline
- PBS pH 7.2
- Tris-HCl/saline buffer 0.05 M Tris-HCl + 0.15 M NaCl
- Tris HCl pH 7.6 (stock solution) Trizima base. D.I. water, dissolve in HCl; adjust to pH 7.6
- DAB 3,3'-Diaminobenzidine tetrahydrochloride
- Tris/saline buffer pH 7.6. Dissolve and aliquot 1 ml into vials and store at -20oC.
- FAB 2 fragments having the following properties are intravenously administered (see Figure 30A): a. Bifunctional antibody with one hypervariable region binding Z2D3 antigen, and the other binding the propeptide of the fibroblast collagenase proenzyme. b. Bifunctional antibody with one hypervariable region binding Z2D3 antigen, and the other binding the propeptide of the neutrophil collagenase proenzyme. c. Bifunctional antibody with one hypervariable region binding Z2D3 antigen, and the other binding the propeptide of the type IV/V collagenase proenzyme. d. Bifunctional antibody with one hypervariable region binding Z2D3 antigen, and the other binding the propeptide of the stromelysin proenzyme. e. A mixture of the four FAB 2 fragments above (a-d), labelled with radionuclide x, and representing a minor component of the overall pool of FAB 2 fragments.
- the patient is scanned with a gamma camera, attuned to radionuclide x, 24 to 48 hours after administration of the FAb 2 fragments, and an estimate is made of the quantity of FAb 2 fragments localized in the target lesions, based on the amount of radiolabelled FAb 2 fragments detected, (see Figure 30B)
- the proenzyme mixture is administered in incremental doses, until the desired amount is localized in the lesions.
- the desired amount is that which will dissolve enough plague ot relieve the arterial obstruction, without causing aneurysm formation or perforation in severely diseased vessels.
- Tissue plasminogen acitivator in intravenously administered in an amount sufficient to generate enough circulating plamsin to cleave the functional enzymes from their bound propeptides, yet insufficient to create a hemorrhagic diathesis, (see Figure 30E)
- collagen type I neutral collagenase
- collagen type III fibroblast collagenase
- collagen type IV/V type IV/V collagenase
- proteoglycans/fibronectin stromelysin
- a representative treatment protocol can be as follows:
- Catheterized artery coronary ostia, carotid, aorta or peripheral vessels.
- Atherosclerosis is characterized by the presence of one or more of the atherosclerotic plaque specific antigens disclosed in the subject invention. Because of the cyclical nature of the immune response, either the plaque antigen or antibody which specifically binds to the antigen may be detected at any one point in time. Accordingly, the presence of either the antigen or antibody thereto is indicative of atherosclerotic plaque.
- Figure 5 shows a comparison of levels of IgA specific to atherosclerotic plaque antigen present in the sera of normal persons less than 35 years of age, and persons diagnosed as having coronary artery disease (CAD).
- CAD coronary artery disease
- Figure 6 shows a comparison of levels of atherosclerotic plague specific antigen present in the sera normal persons greater than 35 years of age, and persons diagnosed as having CAD.
- levels of IgA levels of IgG which specifically binds to the atherosclerotic plaque specific antigen, are higher in persons with CAD.
- 45 of 125 tested showed elevated levels of antigen, whereas only 4 of 25 normal persons under 35 years of age, and 8 of 49 normal persons over 35 years of age showed elevated levels of antigen.
- Figure 7 shows a plot of antigen level vs. patient age for apparently healthy individuals.
- Figure 8 shows the same plot for individuals having 50% or greater occlusion of their coronary artery
- Figure 9 shows the same plot for individuals having mild CAD.
- the amount of antigen present in sera is less dependent upon age than upon severity of CAD. Atherosclerosis is therefore indicated by the presence of the atherosclerotic plaque specific antigen.
- Figure 15 depicts positive prevalence for the antigen in a population based on age. The persons tested were from 31 to 75 years of age.
- the fractions which immediately followed the void volume of the column showed the highest levels of binding to CAD serum, and were used to immunize Balb C mice. Splenocytes from mice that produced antibodies were then fused with the immortal cell line SP2. One such fusion produced hybridoma 15H5.
- the 15H5 monoclonal antibody was then covalently coupled to separose/agarose.
- This solid support antibody complex was then used in a number of assays to determine levels of antigen in various samples. Further, by contacting a PBS extraction of human atherosclerotic plaque with the 15H5 monoclonal antibody-solid support complex, it was possible to remove the auto-antigen from other extracted materials. The resulting complex was then washed and purified auto-antigen was then eluted from the complex.
- Sensitive to periodate treatment yes yes 18. Sensitive to urea no no
- antibodies and antisera which specfically bind to the antigen were reacted with various components found in atherosclerotic plaque (see Table 2).
- the antigen was reacted with various enzymes to determine if the antigen is susceptible to degradation. It was found that proteinases, deoxyribonucleases, lipases, and ribonucleases did not degrade the 15H5 antigen. However, the 15H5 antigen was partially degraded by certain glycosidases, especially ⁇ -amylase, ⁇ -amylase, and glycoamylase. This suggests that the 15H5 antigen comprises a structure which has a carbohydrate nature. The results obtained with individual enzymes are shown in Table 3.
- Phaseolus vulgaris-E - Phaseolus vulgaris-E - (Phytohemaglutinin)
- Sophova japonica - (Pagoda Tree)
- Vicia villosa (Isolectin B 4 ) -
- the antigen was also characterized by hydrolysis using 2M trifluoroacetic acid heated for 104°C for 4 hrs followed by carbohydrate analysis. (Results are shown under Carbohydrate Analysis of Human Plasma Antigen in the Experimental Details Section).
- the atherosclerotic plaque antigen may be characterized by the carbohydrate profile shown in Figures 20-22.
- the antibodies produced by hybridomas 15H5, Z2D3, Q10E7, and 17H3 were tested for binding to various tissues to determine if the antigens are plaque specific. The results of this testing, which was performed using the histological methods described in Procedures for Histology in the Experimental Detail section, are set forth in Table 5.
- Table 6 further characterizes the antibodies by cross reactivity and inhibition of antigens present in CAD serum. Actual inhibition assay results are depicted in Figure 25.
- Figure 26 shows the effect of binding inhibition using monoclonal antibodies instead of CAD serum.
- Coronary artery lesions
- Isotype IgM IgM IgG 1 IgM pI (5.2-5.9) (5.0-5.7) (5.1-5.8) (4.5-5.1)
- Coronary arteries
- figures 31-35 demonstrate the histologically specificity of the the Z2D3 and Q10E7 antigens histologically.
- Figure 31 shows specific binding of the
- FIG. 32 and 33 show specific binding of the Z2D3 antibody in unfixed sections of human coronary artery obtained postmortem. Note that there is little non-specific staining and that in control sections, i.e. those treated with a non-specific monoclonal antibody, there is no staining.
- Figure 34 The three photographs in Figure 34 are from consecutive sections of the artery.
- Figure 34 clearly demonstrates that Z2D3 recognizes a late stage plaque antigen and Q10E7 recognizes an early stage plaque antigen or a normal section of artery.
- Figure 34A shows binding of Z2D3 to be in the area of the occlusion.
- Figure 34B shows binding of Q10E7 to be in the area opposite the area stained by Z2D3, i.e. in an earlier stage of atherosclerosis or normal remaining artery.
- Figure 34C shows that no binding occurs using a non-specific monoclonal antibody.
- Figure 35 demonstrates in vivo binding of 111 In-labeled Z2D3 in rabbit aortas which were treated to produce atherosclerotic plaque. Non-treated sections of the aorta showed only trace amounts of binding.
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Abstract
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US38812989A | 1989-07-31 | 1989-07-31 | |
US388129 | 2003-03-13 |
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EP0485476A1 true EP0485476A1 (fr) | 1992-05-20 |
EP0485476A4 EP0485476A4 (en) | 1993-03-03 |
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Application Number | Title | Priority Date | Filing Date |
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EP19900912266 Withdrawn EP0485476A4 (en) | 1989-07-31 | 1990-07-31 | Atherosclerotic plaque specific antigens, antibodies thereto, and uses thereof |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0485476A4 (fr) |
JP (1) | JPH05500307A (fr) |
AU (1) | AU6164290A (fr) |
CA (1) | CA2060663A1 (fr) |
WO (1) | WO1991002252A1 (fr) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US5776093A (en) | 1985-07-05 | 1998-07-07 | Immunomedics, Inc. | Method for imaging and treating organs and tissues |
US5196324A (en) * | 1989-12-15 | 1993-03-23 | Eli Lilly And Company | Monoclonal antibodies reactive with a human atheroma associated antigen |
AU1159497A (en) * | 1996-11-08 | 1998-06-03 | Regents Of The University Of California, The | Methods and reagents for non-invasive imaging of atherosclerotic plaque |
US6375925B1 (en) * | 1996-11-08 | 2002-04-23 | The Regents Of The University Of California | Methods and reagents for non-invasive imaging of atherosclerotic plaque |
US8129123B2 (en) | 2004-10-05 | 2012-03-06 | The Regents Of The University Of California | Methods for assessing atherogenesis by determining oxidized phospholipid to apolipoprotein B ratios |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0293524A1 (fr) * | 1987-06-02 | 1988-12-07 | Vasocor | Immunoessai de plaque athérosclérotique |
EP0334076A2 (fr) * | 1988-02-04 | 1989-09-27 | Tatsuya Takano | Anticorps monoclonal capable de reconnaître un antigène associé à l'artériosclérose humaine et procédé pour sa préparation |
WO1990005748A1 (fr) * | 1988-11-15 | 1990-05-31 | Massachusetts Institute Of Technology | Proteine receptrice pure et anticorps pour celle-ci |
Family Cites Families (2)
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US4628027A (en) * | 1982-05-19 | 1986-12-09 | Molecular Engineering Associates, Ltd. | Vitro diagnostic methods using monoclonal antibodies against connective tissue proteins |
GB8500918D0 (en) * | 1985-01-15 | 1985-02-20 | Cancer Res Inst | Viral isolates |
-
1990
- 1990-07-31 CA CA002060663A patent/CA2060663A1/fr not_active Abandoned
- 1990-07-31 WO PCT/US1990/004272 patent/WO1991002252A1/fr not_active Application Discontinuation
- 1990-07-31 JP JP2511540A patent/JPH05500307A/ja active Pending
- 1990-07-31 AU AU61642/90A patent/AU6164290A/en not_active Abandoned
- 1990-07-31 EP EP19900912266 patent/EP0485476A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0293524A1 (fr) * | 1987-06-02 | 1988-12-07 | Vasocor | Immunoessai de plaque athérosclérotique |
EP0334076A2 (fr) * | 1988-02-04 | 1989-09-27 | Tatsuya Takano | Anticorps monoclonal capable de reconnaître un antigène associé à l'artériosclérose humaine et procédé pour sa préparation |
WO1990005748A1 (fr) * | 1988-11-15 | 1990-05-31 | Massachusetts Institute Of Technology | Proteine receptrice pure et anticorps pour celle-ci |
Non-Patent Citations (1)
Title |
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See also references of WO9102252A1 * |
Also Published As
Publication number | Publication date |
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WO1991002252A1 (fr) | 1991-02-21 |
AU6164290A (en) | 1991-03-11 |
EP0485476A4 (en) | 1993-03-03 |
CA2060663A1 (fr) | 1991-02-01 |
JPH05500307A (ja) | 1993-01-28 |
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