Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• AAA Abdominal Aortic Aneurysm
• AAA abdominal aortic aneurysm
• Macrophages as well as mesenchymal cells, synthesize many proteinases, including several members of an important family of matrix metalloproteinases (MMPs) that have the capacity to degrade the major connective tissue components: collagen, elastin, fibronectin, laminin, and the proteoglycans.
• MMPs matrix metalloproteinases
• This invention provides a purified human 70 kDa protein, which has been recalculated as 80 kDa based on different standards, and antibodies directed thereto.
• This invention further provides a method of isolating the antibody directed to the human 80 kDa protein using extracts of human abdominal aortic aneurysm.
• This invention provides a method of purifying an 80 kDa protein by affinity chromatography using the isolated antibody directed to the human 80 kDa protein.
• This invention provides a method of determining whether a human subject is predisposed to or has abdominal aortic aneurysm disease by detecting circulating antibody directed to the human 80 kDa protein.
• This invention further provides a method of alleviating aortic aneurysm disease in a human subject by administering the human 80 kDa protein to the subject under conditions that make the human subject tolerant to the human 80 kDa protein or by administering an effective dose of an immunosuppressant drug to the subject.
• FIG. 1A Western Blot. Lanes 1, 2, and 3 represent serial extractions of aortic homogenates from AAA in salt, Brij , and urea buffers, respectively. Native IgG is also represented. The arrow identifies the unique band at -70 kDa which was visualized when the filter was probed with AAA IgG.
• Figure IB Western Blot. Lanes 1, 2, and 3 represent serial extractions of aortic homogenates from normal abdominal aorta in salt, Brij , and urea buffers, respectively. No unique band was seen in this control experiment.
• Figure 2A Immunohistochemistry. Normal aortic section incubated with human sera and purified IgG, respectively, from a non- AAA patient. 2OX magnification.
• Figure 2B Immunohistochemistry. Normal aortic section incubated with human sera and purified IgG, respectively, from a non- AAA patient. 20X magnification.
• Figure 2C Immunohistochemistry. Normal aortic section incubated with human purified AAA IgG. 40X magnification.
• Figure 2D Immunohistochemistry. Normal aortic section incubated with human purified AAA IgG. 4OX magnification.
• This invention provides a purified human 70 kDa protein, which has been recalculated as 80 kDa based on different standards.
• the purified 80 kDa protein is localized to the abdominal aorta and is the antigen recognized by abdominal aortic aneurysm associated imunoglobulins.
• This invention further provides an isolated antibody directed to the 80 kDa protein.
• the isolated antibody directed to the 80 kDa protein may be a polyclonal antibody or a monoclonal antibody.
• the isolated antibody directed to the 80 kDa protein may be purified from a human abdominal aortic aneurysm or it may be serum-derived or monoclonal and prepared using methods well known in the art.
• monoclonal antibodies are prepared using hybridoma technology by fusing antibody producing B cells from immunized animals with myeloma cells and selecting the resulting hybridoma cell line producing the desired antibody.
• Serum derived antibody may be obtained from animals immunized with the 80 kDa protein.
• This invention provides a method of isolating a polyclonal antibody directed to the 80 kDa protein which comprises: a) obtaining a sample of a human abdominal aortic aneurysm; b) preparing an extract of the sample; c) contacting the extract with protein A under conditions allowing the protein A to specifically bind an antibody; and d) isolating the antibody from the protein A.
• the sample of an abdominal aortic aneurysm is obtained using surgical techniques well known to one of skill in the art.
• the sample can be frozen before extraction or homogenized immediately using methods known in the art.
• the extract may be prepared by homogenizing the sample in a salt buffer comprising 0.05 M Tris-HCl and 2 M NaCl, pH 7.5, centrifuging the homogenate at 10,000 g for 1 hour and retaining the supernatant .
• the protein A may be operatively linked to Sepharose for easier separation of the Protein A or Protein A - Immunoglobulin complex from other matter in the extract.
• This invention provides a method of purifying an 80 kDa protein which comprises: a) obtaining a sample of human abdominal aorta; b) preparing an extract of the sample; c) contacting the extract with an antibody directed to the 80 kDa protein under conditions that allow the antibody to specifically bind the 80 kDa protein; and d) purifying the 80 kDa protein from the antibody.
• Methods of antigen purification using isolated antibodies directed to the antigen are well known in the art . Examples of antigen purification include but are not limited to immunoprecipitation and immunoaffinity chromatography (Jacoby, W.B., and Wilchek, M. , 1974; Ausubel, F.M. , et al. 1991; and Harlow, E., and Lane, D. , 1988) .
• This invention provides a method of purifying an 80 kDa protein which comprises: a) obtaining a sample of human aorta; b) preparing an extract of the sample; c) contacting the extract with an antibody directed to the 80 kDa protein operatively linked to Sepharose under conditions that allow the antibody to specifically bind the 80 kDa protein; and d) purifying the 80 kDa protein from the antibody.
• An example of antibodies operatively linked to Sepharose includes but is not limited to an immunoaffinity column. Purification of the antigenic component of the AAA wall (i.e. the 80 kDa protein) can involve the use of an affinity chromatography wherein an affinity column is prepared using the purified IgG's directed to the 80 kDa protein.
• an affinity column capable of purifying 80 kDa protein is purified IgG's directed to the 80 kDa protein linked to CNBR-activated Sepharose 4B (available from Sigma) , wherein the purified 80 kDa protein can be eluted from the column using IM isothiocyanate.
• This invention provides a method of determining whether a human subject is predisposed to or has abdominal aortic aneurysm disease which comprises: a) obtaining a peripheral blood sample from a human subject; b) contacting the blood sample with the purified 80 kDa protein under conditions allowing the protein to specifical7ly bind an antibody directed to the 80 kDa protein; c) detecting the antibody bound to the 80 kDa protein, the detection of the antibody directed to the 80 kDa protein in the peripheral blood sample determines that a human subject is predisposed to or has abdominal aortic aneurysm disease.
• Detection of the antibody bound to the 80 kDa protein includes but is not limited to Enzyme Linked Immunosorbant Assays (Elisa) and Radioimmunoassays.
• Antibody - Antigen binding assays are well known in the art (Harlow, E., and Lane, D. , 1988) . ⁇
• compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
• forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
• a method of alleviating aortic aneurysm disease in a human subject which comprises administering the pharmaceutical composition comprising the 80 kDa protein under conditions that make the human subject tolerant to the human 80 kDa protein.
• Tolerance has been induced in humans to specific autoimmune antigen proteins by administering the antigen orally to the human subject.
• David Trentham, et al. report they have significantly reduced Rheumatoid Arthritis (RA) patients' symptoms by feeding them type II collagen, a protein common in joint cartilage and a possible target of the autoimmune attack in RA (Trentham, D, et al. , 1994) .
• RA Rheumatoid Arthritis
• oral tolerization takes advantage of a trick used by the body through the digestive system suppress immune responses to those proteins instead of triggering them. Oral tolerization attempts to reduce autoimmune attacks by feeding the patients proteins--collagen, in this case--that are found at the site of autoimmune disease and that may have triggered the autoimmunity in the first place (Barinaga, M. , 1994) .
• This invention provides a method of alleviating abdominal aortic aneurysm disease in a human subject which comprises administering an effective amount of an immunosuppressant drug to inhibit autoimmunity.
• immunosuppressant drugs to inhibit autoimmune diseases are known in the art, and examples include the use of cyclophosphamide for patients with Lupus and Methotrexate for patients with Rheumatoid Arthritis.
• IgG and the antigenic component Human IgG is purified from extracts of aneurysm tissue or from normal serum using standard protein A-Sepharose affinity chromatography (available from BioRad) . The IgG preparation is dialyzed against Phosphate Buffer Saline (PBS) and then lyophilized. Purification of the antigenic component of the AAA wall (i.e. the 80 kDa protein) involves the use of an affinity chromatography wherein an affinity column is prepared using the purified IgG's directed to the 80 kDa protein.
• an affinity column capable of purifying 80 kDa protein is purified IgG's directed to the 80 kDa protein linked to CNBR-activated Sepharose 4B
• Immunohistochemistry We have developed methods for fluorescent immunohistochemical detection and color substrate detection of Horseradish Peroxidase (HRP) .
• HRP Horseradish Peroxidase
• the tissues are fixed in paraformaldehyde/lysine/m- periodate, embedded in paraffin, and sectioned. After binding, the first antibody is detected with either: a biotinylated second antibody which is then tagged with streptavidin-HRP and developed with a color reaction; or a fluorescent second antibody.
• Serum and purified IgG from non-AAA patients were used for control experiments. Immunoblotting techniques compared the reactivity of IgG plus secondary antibody versus secondary antibody alone (as a control) against soluble AAA extracts.
• the sections are counterstained with toluidine blue or hematoxylin and eosin, allowing assignment of some of the cell types of interest by morphological criteria.
• We are also taking advantage of the inhibition of secretory processes by monensin to improve detection of the proteinases of interest within the secreting cells. Dual-labelling techniques are also appropriate to this part of the project.
• Protein sequencing The protein for sequencing was run on a 12.5% Laemmli gel and stained in Coumassie Blue R250 in 10% acetic acid/50% methanol followed by destaining in 10% acetic acid/50% methanol for 2 hours. The band of protein is excised from the gel and sequenced directly from the gel slice using N-terminal peptide sequencing.
• the molecular weight of the band representing the protein at -70 kDa has been recalculated based on different standards and -80 kDa is believed to be a better estimate of the molecular weight of the protein.
• AAA disease The role of inflammatory cells as sources of matrix- destructive proteinases in AAA disease is presently under study by our and others (Tilson, et al. 1994) .
• leukocytes We have found leukocytes to be abundant in AAA adventitia.
• AIM-cells AAA- Infiltrating-Monocytes
• a L-cells AAA- Infiltrating-Lymphocytes
• the AIM-cells probably play a significant role in the direct destruction of matrix, and also both AIM- and AIL-cells may interact in signalling to activate mesenchymal cells.
• Recent in- vitro observations suggest that products of AIL-cells in tissue culture can stimulate proteinase production by cultured AIM-cells.
• IgG purified from the wall of AAA specimens is immunoreactive with a protein extractable from aortic matrix with a MW of -70 kDa.
• Immunohistochemical co-localization of the IgG with collagenous bundles in the adventitia is particularly interesting, in view of biomechanical considerations suggesting that the adventitial collagen must fail in the course of aneurysm expansion (Tilson, et al. 1990) .
• the diagnostic implication is that a simple blood test would become possible if the IgG's of interest are circulating.
• the presence of the antibody might be a specific marker for the disease; and the quantity of antibody might be found to reflect the level of activity of the disease. It is even possible that high titers of antibody might predict increase risk for rupture.
• AAA disease has a significant component of autoimmunity not only opens many approaches for a deeper understanding of the underlying genetics and biochemistry but also may lead to new approaches for prevention and treatment .
• RM Greenhalgh JA Mannick
• W.B. Saunders Company London 97-104 (1990) .

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