Background of the Invention

Throughout this application, various publications are referenced in parenthesis by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

Abdominal Aortic Aneurysm (AAA) is one of the top 15 causes of death in humans. Degradation of the extracellular matrix and loss of structural integrity of the aortic wall are features of the abdominal aortic aneurysm (AAA) . The causes of these changes have been the subject of extensive research for more than a decade. Although some recent reports emphasize the importance of conceptualizing a dynamic situation during AAA development, with the simultaneous synthesis of new matrix proteins during the process of enzymatic destruction (Minion, D.J., et al . , 1992; Wang, Y. , et al. , 1992) , changes in the extracellular matrix as a result of proteolytic processes are of central interest during the extensive remodeling of the aorta that occurs during AAA formation. Three recent studies of the histopathologic changes of AAA have suggested that there is a consistent and significant infiltration of inflammatory cells in the adventitial layer (Beckman, E.N., 1986; Brophy, CM., et al. , 1991; Koch, A.E., et al. , 1990) , where much of the collagen of the aorta is localized (Davidson, J.M., et al. , 1985) . Macrophages are prominent in the infiltrate. 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. Brophy, C. M., et al. (1991) demonstrated that routine non-specific abdominal aortic aneurysms (AAA's) have a prominent infiltration of inflammatory cells and that AAA tissues are rich in immunoglobulins. These observations raise the question of whether autoimmune mechanisms play a role in the pathogenesis of AAA development.

Summary of the Invention

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.

Brief Description of the Ficrures

Figure 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.

RECTIFIED SHEET

Detailed Description of the Invention

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. For example, 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. One example of 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) .■

This invention provides a pharmaceutical composition comprising the 80 kDa protein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers. The pharmaceutical composition may be constituted into any form suitable for the mode of administration selected. 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) . Their approach, called 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. The use of 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.

The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follow

thereaf ter .

EXPERIMENTAL DETAILS

Methods:

Purification of 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. One example of 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 BioRad) , wherein the purified 80 kDa protein can be eluted from the column using IM isothiocyanate.

Immunohistochemistry: We have developed methods for fluorescent immunohistochemical detection and color substrate detection of Horseradish Peroxidase (HRP) . 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.

Results:

Our hypothesis that autoantibodies are present in AAA has been tested by purification of immunoglobulins from AAA wall to determine whether they are directed against specific components of the aorta. Immunoglobulin (IgG) , purified from AAA tissue by standard protein A- Sepharose affinity chromatography, was used to detect immunohistochemical reactivity to connective tissue components in sections of normal aorta. The results suggest that the IgG is associated with adventitial collagen (Figures 1A-1D) . Reactivity of IgG from AAA patents codistributed conspicuously with collagenous fibers in the normal aorta on immunohistochemical evaluation, particularly in the adventitia. Control serum and IgG had minimal reactivity with these fibers, and any positive reaction was virtually undetectable. A unique band at ~70 kDa was visualized using Western Blot when the filters were probed with AAA IgG, and no unique bands were detected in the control experiments (Figures 2A and 2B) .

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.

Discussion:

The role of inflammatory cells as sources of matrix- destructive proteinases in AAA disease is presently under study by ourselves and others (Tilson, et al. 1994) . We have found leukocytes to be abundant in AAA adventitia. We refer to these cells as AIM-cells (AAA- Infiltrating-Monocytes) and 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. In addition, we have observed a significant increase in the population of B-cells present by comparison to control atherosclerotic aorta.

In the present work we find 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 present observations have etiologic, diagnostic, and therapeutic implications. From the point of view of etiology, it is conceivable ^' that the mutation predisposing many patients to AAA disease might result in an amino acid sequence alteration in the 80 kDa protein that makes it antigenic. A complete sequence

of this protein might be informative. Of course, there are many other possibilities, such as mutation in a glycoprotein that normally shields the 80 kDa protein from immunological surveillance. Speculation could go on and on, but the present discovery opens new avenues for approaching the identification of one or more new candidate genes.

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.

Finally, the therapeutic implication of this discovery is that, as in rheumatoid arthritis (Trentham, et al. 1993) , a patient made tolerant to the antigenic component would have a decreased risk of progression of AAA disease. It seems self-evident that the discovery that 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 .

References

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