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  Modified proteases that inhibit complement activationUSPTO Application #: 20070093443Title: Modified proteases that inhibit complement activation Abstract: Provided are methods for and compounds for modulating the complement system. In particular, compounds are provided that inhibit complement activation and compounds are provided that promote complement activation. The compounds are therapeutics by virtue of their effects on the complement system. Hence, the compounds that inhibit complement activation can be used for treatment of ischemic and reperfusion disorders, including myocardial infarction and stroke, sepsis, autoimmune diseases, inflammatory diseases and diseases with an inflammatory component, including Alzheimer's Disease and other neurodegenerative disorder. (end of abstract) Agent: Stephanie Seidman Fish & Richardson P.C. - San Diego, CA, US Inventors: Edwin L. Madison, Jack Nguyen, Sandra Waugh Ruggles, Christopher Thanos USPTO Applicaton #: 20070093443 - Class: 514044000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero Ring, Polynucleotide (e.g., Rna, Dna, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20070093443. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] Benefit of priority is claimed to U.S. provisional application Ser. No. 60/729,817, filed Oct. 21, 2005, entitled "MODIFIED PROTEASES THAT INHIBIT COMPLEMENT ACTIVATION," to Edwin Madison. The subject matter of this application is incorporated by reference in it entirety. [0002] This application is related to International PCT application No. (Attorney Docket No. 19049-003WO1/4903PC), filed Oct. 20, 2006, entitled "MODIFIED PROTEASES THAT INHIBIT COMPLEMENT ACTIVATION," to Edwin Madison, Jack Nguyen, Sandra Waugh Ruggles and Christopher Thanos, which also claims priority to U.S. Provisional Application Ser. No. 60/729,817. This application also is related to U.S. application Ser. No. 10/677,977, filed Oct. 02, 2003, entitled Methods of Generating and Screening for Proteases with Altered Specificity; to U.S. application Ser. No. 11/104,110, filed Apr. 12, 2005, entitled Cleavage of VEGF and VEGF Receptor by Wild-Type and Mutant MTSP-1; and to U.S. application Ser. No. 11/104,111, filed Apr. 12, 2005, entitled Cleavage of VEGF and VEGF Receptor by Wild-Type and Mutant Protease. [0003] The subject matter of each of the above-noted applications is incorporated by reference in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ON COMPACT DISCS [0004] An electronic version on compact disc (CD-R) of the Sequence Listing is filed herewith in duplicate (labeled Copy #1 and Copy #2), the contents of which are incorporated by reference in their entirety. The computer-readable file on each of the aforementioned compact discs, created on Oct. 20, 2006, is identical, 2,075 kilobytes in size, and titled 4903SEQ.001.txt. FIELD OF INVENTION [0005] Provided are methods for and compounds for modulating the complement system. In particular, compounds are provided that inhibit complement activation and compounds are provided that promote complement activation. The compounds are therapeutics by virtue of their effects on the complement system. Hence, the compounds that inhibit complement activation can be used for treatment of ischemic and reperfusion disorders, including myocardial infarction and stroke, sepsis, autoimmune diseases, inflammatory diseases and diseases with an inflammatory component, including Alzheimer's Disease and other neurodegenerative disorders. BACKGROUND [0006] The complement (C) system is part of the immune system and plays a role in eliminating invading pathogens and in initiating the inflammatory response. The complement system of humans and other mammals involves more than 30 soluble and membrane-bound proteins that participate in an orderly sequence of reactions resulting in complement activation. The blood complement system has a wide array of functions associated with a broad spectrum of host defense mechanisms including anti-microbial and anti-viral actions. Products derived from the activation of C components include the non-self recognition molecules C3b, C4b and C5b, as well as the anaphylatoxins C3a, C4a and C5a that influence a variety of cellular immune responses. These anaphylatoxins also act as pro-inflammatory agents. [0007] The complement system is composed of an array of enzymes and non-enzymatic proteins and receptors. Complement activation occurs by one of three primary modes known as the "classical" pathway, the "alternative" pathway and the "lectin" pathway (see FIG. 1). These pathways can be distinguished by the process that initiates complement activation. The classical pathway is initiated by antibody-antigen complexes or aggregated forms of immunoglobulins; the alternative pathway is initiated by the recognition of structures on microbial and cell surfaces; and the lectin pathway, which is an antibody-independent pathway, is initiated by the binding of mannan binding lectin (MBL, also designated mannose binding protein) to carbohydrates such as those that are displayed on the surface of bacteria or viruses. Activation of the cascades results in production of complexes involved in proteolysis or cell lysis and peptides involved in opsonization, anaphylaxis and chemotaxis. [0008] The complement cascade, which is a central component of an animal's immune response, is an irreversible cascade. Numerous protein cofactors regulate the process. Inappropriate regulation, typically inappropriate activation, of the process is a facet of or can occur in a variety of disorders that involve inappropriate inflammatory responses, such as those observed in acute and chronic inflammatory diseases. These diseases and disorders include autoimmune diseases, such as rheumatoid arthritis and lupus, cardiac disorders and other inflammatory diseases, such as sepsis and ischemia-reperfusion injury. [0009] Because of the involvement of the complement pathways in a variety of diseases and conditions, components of the complement pathways are targets for therapeutic intervention, particularly for inhibition of the pathway. Examples of such therapeutics include synthetic and natural small molecule therapeutics, antibody inhibitors, and recombinant soluble forms of membrane complement regulators. There are limitations to strategies for preparing such therapeutics. Small molecules have short half-lives in vivo and need to be continually infused to maintain complement inhibition thereby limiting their role, especially in chronic diseases. Therapeutic antibodies result in an immune response in a subject, and thus can lead to complications in treatment, particularly treatments designed to modulate immune responses. Thus, there exists an unmet need for therapeutics for treatment of complement-mediated diseases and diseases in which complement activation plays a role. These include acute and chronic inflammatory diseases. Accordingly, among the objects herein, it is an object to provide such therapeutics to target the activation of the complement cascade and to provide therapeutics and methods of treatment of diseases. SUMMARY [0010] Provided herein are therapeutics and methods that target the activation of the complement cascade and methods of treatment of diseases, including acute and chronic inflammatory diseases. The therapeutics are non-complement proteases that target complement pathway substrates. Included among the non-complement proteases are unmodified proteases that cleave their native substrate as well as a complement substrate and also proteases modified to have increased selectivity or substrate specificity for a target substrate. The modified proteases can exhibit reduced or altered activity with respect to their native substrates. [0011] Among the methods provided herein are methods of modulating complement activation by contacting a non-complement protease with any one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or more target substrates of a complement pathway, whereby a target substrate protein is cleaved such that complement activation in a pathway comprising the target substrate is altered. Uses of proteases for treatment and/or for forumation of medicaments also are provided. Target substrates for these methods and for any of the methods and uses provide herein are complement proteins, including: C1q, C2, C3, iC3, C4, iC4, C5, C6, C7, C8, C9, MBL, Factor B, Factor D, Factor P, MASP-1, MASP-2, C1r, C1s, C4b, C4a, C2b, C2a, C3b, C3a, Ba, Bb and ficolin. Contacting can be effected ex vivo, in vitro and/or in vivo. Exemplary targets include any of those having a sequence of amino acids set forth in any of SEQ ID NOS: 298, 299, 300, 302, 304, 305, 306, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 326, 328, 330, 332, 334, 335, 338, 340, 344, 660-662 and a fragment of any of the targets that exhibits a complement pathway activity, or allelic or species variants thereof or polypeptides having 60, 70, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity. [0012] For all and for any methods and uses provided herein, the target substrates can be present in a body fluid or tissue sample, or can be a collection of target substrates or any other composition containing such substrates. Depending upon the target substrate(s), complement activation can be inhibited or activated. The methods target one or more any complement pathway. Thus, the complement pathway modulated can be selected from among one or more of the classical, alternative and lectin pathways of complement. The non-complement proteases contain modifications at any one or more amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35 or more residues, compared to an unmodified or scaffold protease. The modified amino acid residue(s) increases one or both of specificity for a target substrate or activity towards a target substrate. Exemplary unmodified or scaffold proteases include any one of a serine protease, a cysteine protease, an aspartic protease, a threonine protease and a metallo-protease, such as, for example, granzyme B, granzyme A, granzyme M, cathepsin G, MT-SP1, neutrophil elastase, chymase, alpha-tryptase, beta-trypsase I or II, chymotrypsin, collagenase, factor XII, factor XI, factor CII, factor X, thrombin, protein C, u-plasminogen activator (u-PA), t-plasminogen activator (t-PA), plasmin, plasma kallikrein, chymotrypsin, trypsin, a cathepsin, papain, cruzain, a metalloprotease and allelic variations, isoforms and catalytically active portions thereof. For example, the scaffold protease comprises a sequence of amino acids set forth in any one of SEQ ID NOS: 2, 4, 8, 77, 79, 83, 85, 87, 89, 93, 99, 117, 119, 121, 123, 132, 134, 138, 142, 144, 146, 148, 162, 166, 168, 170, 172, 174, 176, 178, 180, 182, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 218, 220, 222, 224, 226, 238, 248, 250, 260, 262, 280, 282, 373, 375, 377, 379, 381, 383, 385, 387, 547, 549, and 551 and catalytically active portions thereof, allelic and species variants thereof and polypeptides having 60, 70, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity. MT-SP1 or a fragment thereof, such as the polypeptide sequence set forth in SEQ ID NO: 2 and 10, respectively, is exemplary of a scaffold protease. C2 or C3 proteins of a complement pathway(s) are exemplary target substrates of an MT-SP1 protease, modified MT-SP1 protease, or catalytically active portions thereof. [0013] Modification of an MT-SP1 protease or a catalytically active portion thereof include modification(s) at positions 146, 224, 41, and/or 151, based on chymotrypsin numbering. Such modified MT-SP1 proteases include those with any of the following modifications: I41T/Y146D/G151L/K224F, I41T/Y146D/G151L/Q175D/K224F, I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R, AND I41T/Y146D/G151L/Q175D/K224N, I41T/Y146D/G151L/K224N, Y146D/G151L/K224N, I41T/Y146D/G151L/Q175K/K224F, I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F, I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N, I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering. In particular, a modified MT-SP1 contains amino acid modifications I41T/Y146D/G151L/K224F. [0014] The modifications can be in any one or more amino acids that contribute to extended substrate specificity or secondary sites of interaction, such as, for example, modifications in an MT-SP1 protease or a catalytically active portion thereof that correspond to any one or more of amino acid positions 97, 146, 192, and 224 of an MT-SP1 protease, based on chymotrypsin numbering. Exemplary of such modifications are one or more of F97, Y146, Q192, and K224 of the MT-SP1 protease, based on chymotrypsin numbering, such as F97D, F97E, F97A, F97W, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, Q192R, Q192V, K224A, and K224F. Exemplary of such modified MT-SP1 proteases including those polypeptides having a sequence of amino acids set forth in any of SEQ ID NOS: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 14, 38, and 40 and 405-418. Other examples of a modified MT-SP1 protease or a catalytically active portion thereof include amino acid modifications Y146D/K224F or Y146E, such as those corresponding to a modified MT-SP1 polypeptide having a sequence of amino acids as set forth in SEQ ID NOS:12, 404, 28 or 412. [0015] MT-SP1 protease and catalytically active portion thereof include polypeptides that contain one or more of the following modifications: F97N, F97D, F97E, F99Y, F99V, F99W, D217A, D217V, F97A, F97W, F99A, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, W215F, W215Y, Q192V, Q192R, Q192F, K224A, K224F, M180E, Y146D/K224F, D96A, Y146E/K224N, I41T/Y146E/Q175D/K224R, I41T/Y146D/K224F, I41T/Y146E/Q175D/K224N, I41T/Y146E/G151L/Q175D/K224L, Y146E/Q221aE/K224F, I41T/Y146E/G151L/Q175D/K224R, I41T/Y146E/G151L/Q175D/K224N, Q221aD, Y146E/K224R, Y146E/Q175D/K224N, Y146D/K224R, I41T/Y146E/G151L/Q175D/K224F, Y146E/Q175D/K224R, Y146E/L224L, G147E, Y146D/Q175D/K224R, Y146D/Q175L/K224L, Y146D/Q175L/K224L, Y146D/Q175W/K224L, Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A, Y146D/Q175H/K224L, Y146D/Q175Y/K224L, Y146E/K224Y, Y146D/Q175F/K224L, Y146D/Q175F/K225L, Y146D/Q221aL/K224S, I41E/Y146D/K224L, Y146D/D217F/K224L, Y146D/D217F/K224L, H143V/Y146D/K224F, Y146E/K224F, Y146A/K224F, Y146E/K224T, I41T/Y146E/K224L, I41F/Y146D/K224F, I41L/Y146D/K224F, I41T/Y146D/G151L/K224F, I41A/Y146D/K224F, I41E/Y146D/K224F, I41D/Y146D/K224L, I41D/Y146D/K224F, Y146N/K224F, I41T/Y146D/Q175D/K224F, Q192F/K224F, Y146D/Q192A/K224F, Q192V/K224F, I41T/Y146D/Q175D/K224L, I41T/Y146D/Q175D/K224R, I41T/Y146D/Q175D/K224N, I41T/Y146D/G151L/Q175D/K224F, I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R, I41T/Y146D/G151L/Q175D/K224N, I41T/Y146E/Q175D/K224F, I41T/Y146E/Q175D/K224L, I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N, Y146D/Q175D/K224N, Y146D/G151L/K224N, Y146D/Q175R/K224N, Y146D/Q175K/K224N, Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F, I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F, I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N, I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N, and I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering. Exemplary of such modified MT-SP1 proteases, or catalytically active portions thereof, include those polypeptides having a sequence of amino acids set forth in any of SEQ ID NOS: 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40-69, 404-418, 419-447, 524-533, 552-659, or 663-710. In particular, a modified MT-SP1 protease, or a catalytically active portion thereof, is one having a sequence of amino acids set forth in SEQ ID NOS: 596 or 650. [0016] For all methods and uses provided herein, the modification can be selected such that the modified protease, such as MT-SP1, cleaves a substrate recognition site of the target substrate. Target substrate are any of the complement pathway polypeptides noted above and known to those of skill in the art, including for example, C2 and/or C3. For example, where the target substrate is C2 the substrate recognition site includes a sequence of amino acids of SLGR (SEQ ID NO:392). [0017] Other recognition sites targeted in the methods and uses provided herein include a Factor I substrate recognition site, such as LPSR (SEQ ID NO:388), SLLR (SEQ ID NO:389), or HRGR (SEQ ID NO: 390). Modifications in the MT-SP1 protease or a catalytically active portion can correspond to any one or more of amino acid positions 174, 217, 96, 192, 146, or 99 of an MT-SP1 protease, based on chymotrypsin numbering, such as any one or more of amino acids Q174, D217, D96, Q192, Y146, and F99 of a MT-SP1 protease, based on chymotrypsin numbering. Exemplary of such modifications are modifications selected from among one ore more of: Q174H, D217Q, D217N, D217H, D96A, D96V, D96F, D96S, D96T, Q192L, Q192I, Q192F, Y146F, F99A, F99V, F99S, and F99G (see, e.g., the polypeptides having a sequence of amino acids set forth in any of SEQ ID NOS: 41-57 or 419-435. [0018] Another recognition site includes the sequence of amino acids LPSR. Exemplary of a modified protease modified for recogniztion thereof are: an MT-SP1 protease or a catalytically active portion thereof having modifications at sites corresponding to any one or more of amino acid positions 174, 180, 215, 192, or 99 of an MT-SP1 protease, based on chymotrypsin numbering, such as, for example, any one or more of amino acids Q174, M180, W215, Q192, or F99 of a MT-SP1 protease, based on chymotrypsin numbering. Exemplary of such modifications is any one or more of Q174F, Q174V, Q174L, Q174Y, M180E, W215F, W215Y, Q192K, Q192R, Q192Y, and F99Y (see, e.g., modified MT-SP1 polypeptides having a sequence of amino acids as set forth in any of SEQ ID NOS: 36, 58, 59, 61, 62, 69, 416, 436, 437, 439, 440, 447, or 524-533). [0019] Another substrate recognition site for use in the methods herein includes the sequence of amino acids HRGR. Exemplary of a protease with modifications to recognize such sites are an MT-SP1 protease or a catalytically active portion thereof that has modifications that correspond(s) to modifications at any one or more of amino acid positions 215, 174, 217, 192 and 99 of an MT-SP1 protease, based on chymotrypsin numbering, such as, for example, W215, Q174, D217, Q192 and F99 of an MT-SP1 protease, based on chymotrypsin numbering. Exemplary thereof ar modification selected from among: any one or more of W215F, W215Y, Q174A, Q174V, Q174F, Q174R, Q174K, D217A, D217V, Q192E, F99W and F99Y (e.g., an MT-SP1 protease or a catalytically active portion thereof corresponding to a modified MT-SP1 polypeptide containing a sequence of amino acids as set forth in any of SEQ ID NOS: 58-69 and 436-447. Continue reading... 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