Modified proteases that inhibit complement activation

• US 20070093443A1
• Filed: 10/20/2006
• Published: 04/26/2007
• Est. Priority Date: 10/21/2005
• Status: Abandoned Application

• Current Assignee: Vertex Pharmaceuticals Incorporated
• Original Assignee: Vertex Pharmaceuticals Incorporated
• Inventors: Nguyen, Jack, Thanos, Christopher, Madison, Edwin, Ruggles, Sandra
• Application Number:
  US11/584,776
• Publication Number:
  US 20070093443A1
• Time in Patent Office: Days
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 38/482 Serine endopeptidases (3.4.21)
• CPC Class Codes: A61K 38/49 Urokinase; Tissue plasminogen activator
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/6421 from mammals
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)
• CPC Class Codes: C12N 9/6467 : Granzymes, e.g. granzyme A (3.4.21.78); granzyme B (3.4.21.79)
• CPC Class Codes: C12Y 304/21109 : Matriptase (3.4.21.109)

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.

Claims

1. A method of modulating complement activation, comprising contacting a non-complement protease with any one 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.

2. The method of claim 1, wherein contacting the non-complement protease with any one or more target substrates of a complement pathway occurs in vitro.

3. The method of claim 1, wherein contacting the non-complement protease with any one or more target substrates of a complement pathway occurs in vivo.

4. The method of claim 1, wherein contacting the non-complement protease with any one or more target substrates of a complement pathway occurs ex vivo.

5. The method of claim 1, wherein complement activation is inhibited.

6. The method of claim 1, wherein complement activation is increased.

7. The method of claim 1, wherein the complement pathway is selected from among one or more of the classical, alternative and lectin pathways of complement.

8. The method of claim 1, wherein the target substrate is any one or more of 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 and Bb.

9. The method of claim 1, wherein the target substrate comprises a sequence of amino acids set forth in any one 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, and 344, or comprises a fragment thereof that exhibits a complement activity.

10. The method of claim 1 wherein the target substrate is a ficolin.

11. The method of claim 10, wherein the target substrate comprises a sequence of amino acids set forth in any of SEQ ID NOS:

660-662.

12. The method of claim 1, wherein the non-complement protease comprises modifications at any one or more amino acid residues compared to an unmodified or scaffold protease, wherein the modified amino acid residue(s) increases one or both of specificity for a target substrate or activity towards a target substrate.

13. The method of claim 12, wherein the unmodified or scaffold protease is any one of a serine protease, a cysteine protease, an aspartic protease, a threonine protease, or a metallo-protease.

14. The method of claim 13, wherein the scaffold protease is selected from among proteases selected from among 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.

15. The method of claim 14, wherein 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.

16. The method of claim 14, wherein the scaffold protease is an MT-SP1 protease.

17. The method of claim 16, wherein an MT-SP1 protease or a catalytically active portion thereof comprises a sequence of amino acids set forth in SEQ ID NO:

2 or 10, or is an allelic or species variant thereof.

18. The method of claim 16, wherein the target substrate is C2 or C3.

19. The method of claim 16, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from a protease having a modification in position 224 and/or position 146, based on chymotrypsin numbering.

20. The method of claim 18, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from a protease having a modification in position 224 and/or position 146, based on chymotrypsin numbering.

21. The method of claim 16, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from a protease having a modification in position 151, based on chymotrypsin numbering.

22. The method of claim 18, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from a protease having a modification in position 151, based on chymotrypsin numbering.

23. The method of claim 21, wherein the modification in an MT-SP1 protease or catalytically active portion thereof correspond to a protease having a modification selected from among 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.

24. The method of claim 16, wherein the modifications in the MT-SP1 protease or a catalytically active portion thereof are in any one or more amino acids that contribute to extended substrate specificity or secondary sites of interaction.

25. The method of claim 24, wherein the modifications in a MT-SP1 protease or a catalytically active portion thereof correspond to any one or more of amino acid positions 97, 146, 192, and 224 of an MT-SP1 protease, based on chymotrypsin numbering.

26. The method of claim 25, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof correspond to any one or more of amino acids F97, Y146, Q192, and K224 of the MT-SP1 protease, based on chymotrypsin numbering.

27. The method of claim 26, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from any one or more of F97D, F97E, F97A, F97W, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, Q192R, Q192V, K224A, and K224F.

28. The method of claim 16, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof corresponds to a modified MT-SP1 polypeptide having a sequence of amino acids as 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.

29. The method of claim 27, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are Y146D/K224F or Y146E.

30. The method of claim 28, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof correspond to a modified MT-SP1 polypeptide having a sequence of amino acids as set forth in SEQ ID NOS:

596 or 650.

31. The method of claim 16, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from among 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.

32. The method of claim 31, wherein the MT-SP1 protease cleaves a target substrate at a substrate recognition site.

33. The method of claim 32, wherein the target substrate is C2 or C3.

34. The method of claim 33, wherein the target substrate is C2 and the substrate recognition site includes a sequence of amino acids of SLGR (SEQ ID NO:

392).

35. The method of claim 1, wherein the non-complement protease cleaves a substrate recognition site of a target substrate.

36. The method of claim 35, wherein the non-complement protease cleaves a Factor I substrate recognition site.

126. The method of claim 16, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from a protease having a modification in position 41, based on chymotrypsin numbering.

127. The method of claim 18, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from a protease having a modification in position 41, based on chymotrypsin numbering.

37. A method for treating a subject with a complement-mediated disorder, comprising administering a non-complement protease, whereby the non-complement protease cleaves any one or more target substrates of a complement pathway such that complement activation in a pathway comprising the target substrate is altered.

38. The method of claim 37, wherein complement activation is inhibited.

39. The method of claim 38, wherein the inhibition of complement activation leads to a reduction of inflammatory symptoms associated with a complement-mediated disorder selected from among an inflammatory disorder, a neurodegenerative disorder and a cardiovascular disorder.

40. The method of claim 39, wherein the complement-mediated disorder is selected from among sepsis, Rheumatoid arthritis (RA), membranoproliferative glomerulonephritis (MPGN), Multiple Sclerosis (MS), Myasthenia gravis (MG), asthma, inflammatory bowel disease, immune complex (IC)-mediated acute inflammatory tissue injury, Alzheimer'"'"'s Disease (AD), and Ischemia-reperfusion injury.

41. The method of claim 39, wherein the complement-mediated disorder is Guillan-Barre syndrome.

42. The method claim 40, wherein the ischemia-reperfusion injury is caused by an event or treatment selected from among myocardial infarct (MI), stroke, angioplasty, coronary artery bypass graft, cardiopulmonary bypass (CPB), and hemodialysis.

43. The method of claims 37, wherein the complement-mediated disorder results from a treatment of a subject.

44. The method of claim 43, wherein administering a non-complement protease is effected prior to treatment of a subject.

45. The method of claim 37, wherein administering a non-complement protease is effected by contacting a body fluid or tissue sample in vitro, ex vivo, or in vivo with a non-complement protease.

46. The method of claim 43, wherein the treatment results in complement-mediated ischemia-reperfusion injury.

47. The method of claim 46, wherein the treatment is angioplasty or coronary artery bypass graft.

48. The method of claim 37, wherein the complement pathway is one or more of the classical, alternative and lectin pathways.

49. The method of claim 37, wherein the target substrate is any one or more of 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 and Bb.

50. The method of any of claim 37, wherein the target substrate comprises a sequence of amino acids set forth in any one 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, or 344, or comprises a fragment thereof that exhibits a complement activity.

51. The method of claims 37, wherein the target substrate is ficolin.

52. The method of claim 49, wherein the non-complement protease comprises modifications at any one or more amino acid residues compared to an unmodified or scaffold protease, wherein the modified amino acid residue(s) increases one or both of specificity for a target substrate or activity towards a target substrate.

53. The method of claim 52, wherein the unmodified or scaffold protease is any one of a serine protease, cysteine protease, aspartic protease, threonine protease, or metallo-protease.

54. The method of claim 53, wherein the scaffold protease is is selected from among proteases selected from among 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.

55. The method of claim 54, wherein the scaffold protease comprises a sequence of amino acids as 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.

56. The method of claim 54, wherein the scaffold protease is an MT-SP1 protease.

57. The method of claim 56, wherein an MT-SP1 protease or a catalytically active portion thereof comprises a sequence of amino acids set forth in SEQ ID NO:

2 or 10, or is an allelic or species variant thereof.

58. The method of claim 56, wherein the target substrate is C2 or C3.

59. The method of claim 56, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof comprises a modification in position 224, based on chymotrypsin numbering.

60. The method of claim 58, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof comprises a modification in position 224, based on chymotrypsin numbering.

61. The method of claim 56, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof comprises a modification in position 151, based on chymotrypsin numbering.

62. The method of claim 58, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof comprises a modification in position 151, based on chymotrypsin numbering.

63. The method of claim 51, wherein the modification in an MT-SP1 protease or catalytically active portion thereof is selected from among 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.

64. The method of claim 56, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are in any one or more amino acids that contribute to extended substrate specificity or secondary sites of interaction.

65. The method of claim 64, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof correspond to any one or more of amino acid positions 97, 146, 192, and 224 of an MT-SP1 protease, based on chymotrypsin numbering.

66. The method of claim 65, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof correspond to any one or more of amino acids F97, Y146, Q192, and K224 of the MT-SP1 protease, based on chymotrypsin numbering.

67. The method of claim 66, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from any one or more of F97D, F97E, F97A, F97W, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, Q192R, Q192V, K224A, and K224F.

68. The method of claim 56, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof corresponds to a modified MT-SP1 polypeptide having a sequence of amino acids as 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.

69. The method of claim 67, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are Y146D/K224F or Y146E.

70. The method of claim 68, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof correspond to a modified MT-SP1 polypeptide having a sequence of amino acids as set forth in SEQ ID NO:

596 or 650.

71. The method of claim 56, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from among 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.

72. The method of claim 71, wherein the MT-SP1 protease cleaves a substrate recognition site of the target substrate.

73. The method of claim 72, wherein the target substrate is C2 or C3.

74. The method of claim 73, wherein the target substrate is C2 and the substrate recognition site includes a sequence of amino acids of SLGR (SEQ ID NO:

392).

75. The method of claim 37, wherein the non-complement protease cleaves a substrate recognition site of the target substrate.

76. The method of claim 75, wherein the non-complement protease cleaves a Factor I substrate recognition site.

77. The method of claim 37, wherein the non-complement protease or a catalytically active portion thereof is administered in combination with a second agent for treating a complement-mediated disorder.

78. The method of claim 77, wherein the second agent is an anti-inflammatory agent or an anticoagulant.

79. The method of claim 78, wherein the second agent is selected from among any one or more of a NSAID, antimetabolite, corticosteroid, analgesic, cytotoxic agent, pro-inflammatory cytokine inhibitor, anti-inflammatory cytokines, B cell targeting agents, compounds targeting T antigens, adhesion molecule blockers, chemokines receptor antagonists, kinase inhibitors, PPAR-γ

ligands, complement inhibitors, heparin, warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate, argatroban, lepirudin, bivalirudin, and ximelagatran.

128. The method of claim 56, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof comprises a modification in position 41, based on chymotrypsin numbering.

129. The method of claim 58, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof comprises a modification in position 41, based on chymotrypsin numbering.

80. A combination, comprising:

(a) a non-complement protease that cleaves any one or more complement target substrates of a complement pathway such that complement activation in a pathway comprising the target substrate is altered; and

(b) a second agent or agents for treating a complement-mediated disorder.

81. The combination of claim 80, wherein the second agent or agents is an anti-inflammatory agent(s) or anticoagulant(s).

82. The combination of claim 81 wherein, the anti-inflammatory agent(s) is selected from among any one or more of a NSAID, antimetabolite, corticosteroid, analgesic, cytotoxic agent, pro-inflammatory cytokine inhibitor, anti-inflammatory cytokines, B cell targeting agents, compounds targeting T antigens, adhesion molecule blockers, chemokines receptor antagonists, kinase inhibitors, PPAR-γ

ligands, complement inhibitors, heparin, warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate, argatroban, lepirudin, bivalirudin, and ximelagatran.

83. The combination of claim 80, wherein complement activation is inhibited.

84. A modified non-complement protease, comprising modifications in any one or more amino acids of a scaffold protease, wherein:

the modified amino acid residue(s) increases one or both of specificity for a target substrate or activity towards a target substrate, wherein the target substrate is a complement protein.

85. The modified non-complement protease of claim 84, wherein the target substrate is any one or more of 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, or Bb.

86. The modified non-complement protease of claim 84, wherein the target substrate comprises a sequence of amino acids set forth in any one 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, and 344, or comprises a fragment thereof that exhibits a complement activity.

87. The modified non-complement protease of claim 84, wherein the target substrate is a ficolin.

88. The modified non-complement protease of claim 87, wherein the target substrate comprises a sequence of amino acids set forth in any of SEQ ID NOS:

660-662.

89. The modified non-complement protease of claim 84, wherein the scaffold protease is selected from among a serine protease, cysteine protease, aspartic protease, threonine protease, and metallo-protease.

90. The modified non-complement protease of claim 89, wherein the scaffold protease is selected from among 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.

91. The modified non-complement protease of claim 90, wherein the scaffold protease comprises a sequence of amino acids as 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.

92. The modified non-complement protease of claim 90, wherein the scaffold protease is an MT-SP1 protease.

93. The modified non-complement protease of claim 92, wherein the MT-SP1 protease or a catalytically active portion thereof comprises a sequence of amino acids set forth in SEQ ID NO:

2 or 10.

94. The modified non-complement protease of claims 92, wherein the target substrate is C2 or C3.

95. The modified non-complement protease of claim 92, comprising at least two or more modifications, wherein one modification is at position 146 and the second modification is at position 224, based on chymotrypsin numbering, provided that:

(i) where the protease includes only two modifications, the protease does not include Y146D and K224F as the two modification; and

(ii) where the protease contains three modifications, the protease does not include F99V or I or L or T with Y146D and K224F.

96. The modified non-complement protease of claim 94, comprising at least two or more modifications, wherein one modification is a position 146 and the second is at position 224, based on chymotrypsin numbering, provided that:

(i) where the protease includes only two modifications, the protease does not include Y146D and K224F as the two modification; and

(ii) where the protease contains three modifications, the protease does not include F99V or I or L or T with Y146D and K224F.

97. The modified non-complement protease of claim 92, comprising at least one modification at position 151, based on chymotrypsin numbering.

98. The modified non-complement protease of claim 94, comprising at least one modification at position 151, based on chymotrypsin numbering.

99. The modified non-complement protease of claim 97, wherein modification(s) in an MT-SP1 or catalytically active portion are selected from among 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.

100. The modified non-complement protease of claim 99, wherein modification(s) in an MT-SP1 or catalytically active portion correspond to modification of I41T/I46D/G151L/K224F, based on chymotrypsin numbering.

101. The modified non-complement protease of claim 92, wherein modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from any one or more of D96A, D96V, D96F, D96F, D96S, D96T, F99S, F99G, Q174H, Q174A, Q174V, Q174F, Q174R, Q174K, Q174L, Q174Y, Q192L, Q192I, Q192E, Q192K, Q192Y, D217Q, D217N, D217H, K224A, based on chymotrypsin numbering.

102. The modified non-complement protease of claim 92, wherein modification(s) in an MT-SP1 protease or a catalytically active portion thereof are selected from among 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, and 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.

103. The modified non-complement protease of claim 92, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof correspond to a modified MT-SP1 polypeptide having a sequence of amino acids as set forth in any one of SEQ ID NOS:

41-51, 56, 57, 60-64, 67, 69, 419-429, 431, 434, 435, 438-442, 445, 524, 525, 527-530, 532, 533, 552-659, or 663-710.

104. The modified non-complement protease of claim 92, wherein the modification(s) in an MT-SP1 protease or a catalytically active portion thereof correspond to a modified MT-SP1 polypeptide having a sequence of amino acids as set forth in any one of SEQ ID NOS:

596 or 650.

105. The modified non-complement protease of claim 103, wherein the MT-SP1 protease cleaves a substrate recognition site of the target substrate.

106. The modified non-complement protease of claim 105, wherein the target substrate is C2 or C3.

107. The modified non-complement protease of claim 106, wherein the target substrate is C2 and the substrate recognition site includes a sequence of amino acids of SLGR (SEQ ID NO:

392).

108. A pharmaceutical composition, comprising a modified non-complement protease of claim 84.

109. A combination, comprising:

(a) a pharmaceutical composition of claim 108;

and

(b) a second agent or agents for treating a complement-mediated disorder.

110. The pharmaceutical composition of claim 108, further comprising a pharmaceutically acceptable excipient.

112. A kit, comprising the pharmaceutical composition of claim 108, a device for administration of the composition and, optionally, instructions for administration.

113. A nucleic acid molecule, comprising a sequence of nucleotides that encodes any one of the modified non-complement proteases of claim 84.

114. A vector, comprising the nucleic acid molecule of claim 113.

115. A cell, comprising the vector of claim 114.

116. A method of treatment, comprising administering to a subject a nucleic acid molecule of claim 113.

117. The method of treatment of claim 116, wherein the nucleic acid molecule is introduced into a vector for administration.

118. The method of treatment of claim 117, wherein the vector is an expression vector.

119. The method of treatment of claim 118, wherein the vector is episomal.

120. The method of treatment of claim 117, wherein the expression vector is selected from among an adenovirus vector, an adeno-associated virus vector, EBV, SV40, cytomegalovirus vector, vaccinia virus vector, herpesvirus vector, a retrovirus vector, a lentivirus vector, or an artificial chromosome.

121. The method of treatment of claims 117, wherein the nucleic acid is administered in vivo or ex vivo.

122. The method of treatment of claim 121, wherein ex vivo treatment comprises administering the nucleic acid into a cell in vitro, followed by administration of the cell into the subject.

123. The method of treatment of claim 122, wherein the cell is from a suitable donor or from the subject to be treated.

124. The method of treatment of claim 116, wherein the subject is a human.

125. A fusion protein, comprising a catalytically active portion of a protease of claim 84 that is fused to a non-protease polypeptide.

130. The modified non-complement protease of claim 92, comprising at least one modification at position 41, based on chymotrypsin numbering.

131. The modified non-complement protease of claim 94, comprising at least one modification at position 41, based on chymotrypsin numbering.

111. The pharmaceutical composition of 110, wherein the pharmaceutical composition is formulated for systemic, oral, nasal, pulmonary, local, or topical administration.