Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 11 to 14 amino acid residues in defined sequence
Reexamination Certificate
1999-12-27
2001-08-21
Nolan, Patrick (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
11 to 14 amino acid residues in defined sequence
C530S328000, C530S329000, C530S330000, C530S331000, C435S810000
Reexamination Certificate
active
06277955
ABSTRACT:
BACKGROUND OF THE INVENTION
Autoimmune diseases are characterized by an abnormal immune response (involving either immune system cells or antibodies) directed against normal autologous (self) tissues. Autoimmune diseases afflict huge numbers of individuals throughout the world.
A normal immune system has the capacity to identify and destroy a large variety of foreign invader organisms such as bacteria and viruses. Remarkably, a normal immune system can readily distinguish foreign substances from self, and thereby is able to react vigorously against potentially pathogenic entities from the environment without harming the host's own cells.
The immune system's non-reactivity to self is termed immunological tolerance. In pathological situations, immunological tolerance to a wide variety of self substances is broken, resulting in an autoimmune response to self. If of an appropriate nature and of sufficient severity and duration, the anti-self response will result in an autoimmune disease. In certain autoimmune diseases, specific elements of the immune system predominate in mediating the pathogenic process, while in other autoimmune diseases, all of the components of the immune system cooperate to produce disease. Antibodies are considered to play the major causal roles in diseases such as systemic lupus erythematosus, myasthenia gravis and Graves'disease, while cellular immune mechanisms are believed to be those primarily involved in multiple sclerosis (MS) and insulin dependent diabetes (IDD).
A number of strategies have been used or proposed to suppress autoimmune diseases, most notably drugs, such as cyclophosphamide, cyclosporin A, methotrexate, and Imuran (azathioprine). Steroid compounds, such as prednisone and methylprednisolone, are also employed in many instances. These drugs have limited long term efficacy against both cell- and antibody-mediated autoimmune diseases. Use of such drugs is limited by virtue of their toxic side effects which include “global” immunosuppression. Prolonged treatment with these drugs inhibits the normal protective immune response to pathogenic microorganisms, thereby increasing the risk of infections. A further drawback is that immune-mediated elimination of aberrant cells is impaired and there is, thus, an increased risk that malignancies will develop in patients receiving prolonged global immunosuppression.
Known autoimmune disorders include diabetes, multiple sclerosis, autoimmune uveitis, rheumatoid arthritis, Addison's disease, thyroiditis, atrophic gastritis, myasthenia gravis, idiopathic thrombocytopenic purpura, hemolytic anemia, systemic lupus erythematosus, primary biliary cirrhosis, Wegener's granulomatosis,polyarteritisnodosa, and inflammatory bowel disease.
Wegener's granulomatosis (WG) is characterized by necrotizing granulomatous lesions of the respiratory tract, glomerulonephritis,and, frequently, vasculitis involving other organs. WG is known to be rare, but the exact annual incidence of WG in the general population is not known. Although the incidence of WG is quite low, the syndrome has been observed in persons aged 3 months to 75 years; the peak incidence is in the fourth and fifth decades.
The exact cause of WG is unknown. The clinical course of WG, with initial respiratory tract involvement and then later glomerulonephritis, suggests a possible chain of events in which a pathogenic agent gains entry to the respiratory tract and elicits an inflammatory response that later extends to other tissues.
The differentiation of Wegener's granulomatosis from those diseases that present with similar histopathologic findings is important because management may be different. Infections, especially those with Mycobacterium, Nocardia, or fungi, must be ruled out.
In the early 1980s, researchers described the presence of antibodies directed against cytoplasmic components of neutrophils in patients with segmental necrotizing glomerulonephritis and systemic vasculitis. The presence of these anticytoplasmic antibodies in WG has been reported. These antibodies in the serum of patients with WG cause a characteristic cytoplasmic granular staining pattern by immunofluorescence. These antibodies are called antineutrophil cytoplasmic antibodies (ANCAs). The term c-ANCA is used for cytoplasmic staining antineutrophil cytoplasmic antibody, and p-ANCA for perinuclear staining antineutrophil cytoplasmic antibody.
ANCAs are important in the diagnosis of patients with necrotizing, granulomatous vasculitis, particularly Wegener's granulomatosis (WG). The specificity c-ANCAs is high for the presence of WG (up to 90%). c-ANCAs are directed against proteinase-3 (PR-3), a serine proteinase, found in azurophilic granules of human neutrophils. PR-3 consists of 228 amino acids and has a molecular weight of 29 kD. The most utilized method for detection of ANCAs is indirect immunofluorescence (IIF) using ethanol-fixated human neutrophils as substrate. However, a significant number of false positive and false negative results have been described with this method. In order to achieve greater diagnostic specificity, anti-PR-3 ELISAs have been utilized. However, all extraction procedures for PR-3 from azurophilic granules, including reverse HPLC, have been troubled with impurities, particularly lactoferrin, which make PR-3 ELISAs less specific. Also, it is relatively difficult and expensive to purify PR-3; therefore, an alternative method for detecting the autoantibodies would be helpful.
The amino acid sequence of the PR-3 protein is known and attempts have been made to produce it recombinantly. See, for example, Campanelli, D., M. Melchior, Y. Fu, M. Nakata, H. Shuman, C. Nathan, J. E. Gabay (1990)
J. Exp. Med
172:1709 -1715. So far, the recombinantly produced protein has not performed satisfactorily in the assays for WG. Another problem that is inherent in the use of the full-length PR-3 protein is that this protein is a highly active protease which degrades other proteins as well as itself. The strong protease activity of this protein makes it difficult to work with.
BRIEF SUMMARY OF THE INVENTION
The subject invention pertains to the identification of peptides which are useful in an assay for detecting the autoimmune disease known as Wegener's granulomatosis (WG).
Specifically, the subject invention pertains to peptide fragments of the full-length PR-3 protein which can be used in an assay to detect autoantibodies associated with WG. These peptides are easy and inexpensive to manufacture and are, therefore, advantageous compared to the current use of the full-length purified PR-3 protein. These peptides can also be used in the treatment of WG.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO. 1 is synthetic peptide PR3-5 according to the subject invention.
SEQ ID NO. 2 is synthetic peptide PR3-8 according to the subject invention.
SEQ ID NO. 3 is synthetic peptide PR3-11 according to the subject invention.
DETAILED DISCLOSURE OF THE INVENTION
The subject invention concerns the identification of PR-3 peptides which react with high sensitivity with c-ANCA-positive sera from patients with WG. Three peptides, each 11 amino acids long, are specifically exemplified herein. These peptides have been synthesized in our protein core laboratory. The purity of these PR-3 peptides was found to be >99% by HPLC.
The identified peptides are:
PR3-5: Ile-Cys-Asp-Gly-Ile-Ile-Gln-Gly-Ile-Asp-Ser (SEQ ID NO. 1)
PR3-8: Ala-His-Cys-Leu-Arg-Asp-Ile-Pro-Gln-Arg-Leu (SEQ ID NO. 2)
PR3-1: Leu-Arg-Asp-Ile-Pro-Gln-Arg-Leu-Val-Asn-Val (SEQ ID NO. 3)
Those skilled in the art, having the benefit of the instant disclosure, could make minor modifications to the exemplified peptides without departing from the scope of the instant invention. Such minor modifications would include, for example, adding a small number of amino acids, corresponding to the PR-3 protein, to one or both ends of the peptides.
The peptides of the subject invention can be used in standard assays to detect the onset, or monitor the progression of, WG. Also, as described herein, th
Nolan Patrick
Saliwanchik Lloyd & Saliwanchik
University of Florida
LandOfFree
Materials and method for the detection and treatment of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Materials and method for the detection and treatment of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Materials and method for the detection and treatment of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2550058