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  Peptide and polypeptide inhibitors of complement c1sUSPTO Application #: 20050267035Title: Peptide and polypeptide inhibitors of complement c1s Abstract: The complement system plays an important role in providing resistance to infections and in the pathogenesis of tissue injury. Yet an inappropriate activation of complement can result in a variety of disorders. The present invention provides C1s catalytic site-directed moieties, C1s exosite binding moieties, and bivalent polypeptide inhibitors comprising such moieties, which can be used to treat conditions characterized by inappropriate complement activation. (end of abstract) Agent: Michelle L. Lewis Patent Department - Seattle, WA, US Inventors: Robert R. West, Paul O. Sheppard, Brian A. Fox USPTO Applicaton #: 20050267035 - Class: 514013000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 16 To 24 Peptide Repeating Units In Known Peptide Chain The Patent Description & Claims data below is from USPTO Patent Application 20050267035. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional application No. 60/212,998. filed Jun. 21, 2000, and 09/883,727, filed Jun. 18, 2001, the contents of which are incorporated by reference. TECHNICAL FIELD [0002] The present invention relates generally to new peptides and polypeptides, which inhibit complement C1s. BACKGROUND OF THE INVENTION [0003] The complement system is considered an ancient part of the immune system, which serves to discriminate self and non-self (see, for example, Rother et al. (Eds.), The Complement System, Second Edition (Springer-Verlag 1998), Morely and Walport, The Complement Factsbook (Academic Press 1999), and Morgan (Ed.), Complement Methods and Protocols (Humana Press, Inc. 2000)). Although the complement system plays an important role in providing resistance to infections, an inappropriate activation of complement can result in a variety of disorders. [0004] There are two main pathways for complement activation, which are known as the classical and alternative pathways. Both pathways comprise a cascade of enzyme activation, which leads to the production of a terminal membrane attack complex that targets immune complexes or microorganisms. The alternative pathway is activated by the chance binding of C3b with the surface of a microorganism. The classical pathway is the principal antibody-directed mechanism for the activation of complement. C1, the first enzyme complex in the classical pathway, is a pentamolecular complex consisting of a single C1q molecule, and two C1r and C1s molecules. In the classical pathway, an antibody binds with C1q, which causes the activation of the C1r molecules. These activated proteins then cleave the C1s molecules to form active C1s serine proteases, which act on the next two components of the classical complement pathway, C4 and C2. Cleaved portions of these complement proteins, known as C4b and C2a, then form C3 convertase, which goes on to cleave the next component in the cascade, C3. Thus, C1s plays a key role, because one C1s molecule can generate multiple C4b molecules, which have an amplification effect on the system. [0005] Molecules that inhibit complement may be beneficial for treatment of diseases in which complement activation has been shown to occur, such as adult respiratory distress syndrome, ischemia-reperfusion injury (myocardial infarct, stroke, skeletal muscle, lung inflammation), hyperacute rejection (transplantation), sepsis, cardiopulmonary bypass, burns, wound healing, asthma, restenosis, multiple organ dysfunction syndrome, trauma, hemorrhagic shock, Guillain-Barre syndrome, paroxysmal nocturnal hemoglobinuria, glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, infertility, Alzheimer's disease, organ rejection, myasthenia gravis, multiple sclerosis, platelet storage, serum sickness, various hemolytic anemias, and hemodialysis See, for example, Vogt, Trends Pharm. Sci. 6:114 (1985), and Makrides, Pharm. Rev. 50:59 (1998). [0006] Many different types of compounds have been found to be inhibitors of classical complement, including diamines, amino acids and their derivatives, polynucleotides, polyanions, pyridinium sulphonylfluorides and phenothiazines (see, for example, Ashghar, Pharmac. Rev. 36:223 (1984)). Peptide inhibitors are exemplified by amino acid sequences that mimic the C1 fixing sequences of IgG, glutathione, and leupeptin (see, for example, Boackle et al., Nature 282:742 (1979); Takada et al., Immunology 34:509 (1979)). A tripeptide based on C-terminal sequences of C3a and C5a has been shown to be a substrate for C4b2a, CVFBb and C1s, while substrate-like inhibitors of C3 convertase have also been prepared (see, for example, Andreatta et al., In Enzyme Inhibitors, Brodbeck (Ed.), pages 261-272 (1981); Caporale et al., J. Immun. 126:1963 (1981)). [0007] Few compounds have been found to inhibit the alternative pathway. Complestatin, a microbial product believed to bind to factor B is one example of such an inhibitory compound (Kaneko et al., J. Immun. 124:1194 (1980)). Many inhibitors described above require relatively high concentrations, and lack specificity. [0008] Protein inhibitors of complement have been described more recently, and include: soluble complement receptor (sCRI), a humanized monoclonal antibody to C5, and BD001, a recently described protein derived from a leech, which inhibits C1s (Liszewski and Atkinson, Exp Opin, Invest. Drugs 7:323 (1998); Seale and Finney, International Publication No. WO99/36439). Seale and Finney reported that BD001 has the following amino acid sequence: 1 (SEQ ID NO:1) AKKKLPKCQK QEDCGSWDLK CNNVTKKCEC RNQVCGRGCP KERYQRDKYG CRKCLCKGCD GFKCRLGCTY GFKTDKKGCE AFCTCNTKET ACVNIWCTDP YKCNPESGRC EDPNEEYEYD YE. [0009] The discovery of new C1s-inhibitory peptides and polypeptides fulfills a need in the art by providing new compositions useful in diagnosis and therapy. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein. BRIEF SUMMARY OF THE INVENTION [0010] The present invention provides novel peptides and polypeptides that can inhibit the complement system. The present invention also provides methods of producing these peptides and polypeptides. DESCRIPTION OF THE INVENTION [0011] 1. Overview [0012] BD001, a leech protein that inhibits C1s and factor XII activation, was produced in two separate expression systems: baculovirus and Pichia methanolica. The baculovirus system produced a protein that is virtually identical to the native inhibitory protein isolated from the salivary complexes of the leech. That is, mass spectroscopy and N-terminal sequencing indicated that the baculorivus-derived material had the correct stop and start sites, N-glycosylation at the expected position, and tyrosine sulfation at the C-terminus on three tyrosine residues. Moreover, bioassays of this expressed material in an isolated enzymatic assay of C1s showed equivalence with the native leech material. [0013] In contrast, the protein expressed in Pichia methanolica lacked the tyrosine sulfation at the C-terminus, and N-glycosylation. In addition, a portion of Pichia-produced BD001 molecules were truncated at the C-terminus. This Pichia material was found to be about 10 fold less active in bioassays, compared with the baculorivus-expressed material. Computer modeling of C1s and other serine proteases indicated that alterations in the C-terminus of BD001 could lead to diminished binding with the exosite region of C1s. This result is consistent with the decreased activity of BD001 that contained variations in the C-terminus. [0014] Further sequence analyses revealed that complement protein C4, which is the endogenous substrate for C1s, includes a region that has a string of anionic residues with three tyrosine residues in a similar arrangement to the amino acid sequence of BD001. In addition, studies have shown that these three tyrosine residues of C4 must be sulfated in order for this molecule to have activity (Hortin et al., J. Biol. Chem. 261:1786 (1986); Hortin et al., Proc. Nat'l Acad. Sci. USA. 86:1338 (1989)). Studies described herein substantiate the importance of tyrosine sulfation for BD001 activity. [0015] Without being bound by theory, these collective observations indicate that the C-terminal portion of BD001 mimics a portion of C4 to confer specificity of binding of BD001 for C1s. Accordingly, small peptide or peptidomimetic inhibitors of C1s can be devised, which are based upon the BD001 sequence. [0016] As described herein, the present invention provides complement C1s inhibitors useful as therapeutic agents. These inhibitory peptides and polypeptides are also useful as preservatives in blood samples. In addition, peptides and polypeptides described herein can be used in affinity purification procedures to isolate C1s. [0017] In particular, the present invention provides polypeptides that inhibit complement C1s, wherein the polypeptides are characterized by the formula: "P-N-[DE](2)-[YX.sub.1X.sub.2X.sub.3]-[DE](1,2)-[YX.sub.1X.sub.2- X.sub.3]-[DE]-[YX.sub.1X.sub.2X.sub.3]-[DE](1,2)," where amino acid residues in square brackets indicate acceptable amino acids, numbers in parentheses indicate the number of amino acid residues, "X.sub.1" represents Phe-(p-CH.sub.2)SO.sub.3H, "X.sub.2" represents sulfated tyrosine, and "X.sub.3" represents 2-sulfotyrosine (SEQ ID NO:127). Suitable polypeptides include polypeptides characterized by the formula: "P-N-[DE](2)-[YX.sub.1X.sub.2X.sub.3]-[DE](1,2)-[YX.sub.1X.sub.2X.sub.3]-- [DE]-[YX.sub.1X.sub.2X.sub.3]-[DE]" (SEQ ID NO:129), the formula: "P-N-E-E-[YX.sub.1X.sub.2X.sub.3]-E-[YX.sub.1X.sub.2X.sub.3]-E-[YX.sub.1X- .sub.2X.sub.3]-E" (SEQ ID NO:130), or by the amino acid sequence: "PNEEY EYEYE" (SEQ ID NO:125). [0018] The present invention also proves polypeptides that inhibit complement C1s, wherein the polypeptide comprise an amino acid sequence that is characterized by the formula: "[AP]-N-[DE](2)-[X.sub.1X.sub.2X.su- b.3]-[DE](2)-[X.sub.1X.sub.2X.sub.3]-[DE]-[X.sub.1X.sub.2X.sub.3]-[DE](1,2- )," where amino acid residues in square brackets indicate acceptable amino acids, numbers in parentheses indicate the number of amino acid residues, "X.sub.1" represents Phe-(p-CH.sub.2)SO.sub.3H, "X.sub.2" represents sulfated tyrosine, and "X.sub.3" represents 2-sulfotyrosine (SEQ ID NO:126). Additional examples of complement C1s inhibitors include a peptide or polypeptide that inhibits complement C1s, wherein the peptide or polypeptide comprises the amino acid sequence "CRLGC" (amino acid residues 64 to 68 of SEQ ID NO:1), and wherein the peptide or polypeptide consists of five to thirty amino acid residues. For example, a suitable polypeptide consists of the amino acid sequence: "GCDGFKCRLG CTYGFKTDKK GCEAFCTCNT" (SEQ ID NO:53), whereas a suitable peptide consists of the amino acid sequence: "CRLGC." Continue reading... Full patent description for Peptide and polypeptide inhibitors of complement c1s Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Peptide and polypeptide inhibitors of complement c1s patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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