Masp-3, a complement-fixing enzyme, and uses for it

• US 20030186419A1
• Filed: 02/14/2003
• Published: 10/02/2003
• Est. Priority Date: 12/02/1999
• Status: Abandoned Application

• Current Assignee: Jens Christian Jensenius
• Original Assignee: Jens Christian Jensenius
• Inventors: Jensenius, Jens Christian
• Application Number:
  US10/148,671
• Publication Number:
  US 20030186419A1
• Time in Patent Office: Days
• US Class Current: 435/226
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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: C12N 9/6424 Serine endopeptidases (3.4.21)

Abstract

The invention relates to the discovery and characterization of mannan binding lectin-associated serine protease 3 (MASP-3), a new serine protease that acts in the MBLectin complement fixation pathway.

Claims

1. A substantially pure mannan-binding lectin associated serine protease-3 (MASP-3) polypeptide, wherein said polypeptide comprises either

i) an amino acid sequence identified as SEQ ID NO 5 or a functional equivalent thereof comprising an amino acid sequence at least 85% identical to SEQ ID NO 5;

or

ii) an amino acid sequence identified as SEQ ID NO 1 or a functional equivalent thereof comprising an amino acid sequence at least 85% identical to SEQ ID NO 1;

or

iii) an amino acid sequence identified as SEQ ID NO 2 or a functional equivalent thereof comprising an amino acid sequence at least 50% identical to SEQ ID NO 2.

2. The polypeptide according to claim 1, said polypeptide being conjugated to a label or toxin.

3. The polypeptide according to any of the preceding claims, having a molecular mass of about 110 kDa under non-reducing conditions on an SDS-PAGE.

4. The polypeptide according to claim 3, said polypeptide containing the sequence Identified as SEQ ID NO 5.

5. The polypeptide according to any of the preceding claims, having a molecular mass of about 48 kDa under reducing conditions on an SDS-PAGE.

6. The polypeptide according to claim 5, said polypeptide containing the sequence identified as SEQ ID NO 5.

7. The polypeptide according to claim 1, said polypeptide having serine protease activity.

8. The polypeptide according to any of the preceding claims, said polypeptide being capable of MASP-3 activity in an in vitro assay for MBL pathway of complement function.

9. The polypeptide according to any of the preceding claims, said polypeptide being capable of competitively inhibiting MASP-3 serine protease activity.

10. The polypeptide according to claim 1 or a polypeptide comprising a fragment of the polypeptide of SEQ ID NO:

1, SEQ ID NO;

2 or SEQ ID NO;

5, said polypeptide being a competitive inhibitor of complexing of MBL/MASP-3.

11. An Isolated nucleic acid molecule encoding the polypeptide of any of the claims 1 to 10, the molecule comprising a nucleotide sequence encoding a polypeptide having sequence that is at least 50% identical to the sequence of SEQ ID NO:

1 or 2, or at least 85% identical to the sequence of SEQ ID NO;

5.

12. The isolated nucleic acid sequence according to claim 11, encoding a mannan-binding lectin associated serine protease-3 (MASP-3), wherein the nucleic acid comprises a sequence at least 85% identical to SEQ ID NO:

3.

13. The isolated nucleic acid sequence according to claim 11, encoding a mannan-binding lectin associated serine protease-3 (MASP-3), said nucleic acid sequence being at least 85% identical to SEQ ID NO:

4.

14. A nucleic acid vector comprising the nucleic acid molecule of any of the claims 11 to 13.

15. The nucleic acid vector of claim 14, wherein said vector is an expression vector.

16. The vector of claim 15, further comprising a regulatory element.

17. A cell comprising a vector as defined in any of claims 14 to 16.

18. A cell comprising a nucleic acid sequence as defined in any of claims 11 to 13.

19. The cell according to any of claims 17 to 18 being selected from a yeast cell, or a bacteria cell.

20. An antibody produced by administering a MASP-3 polypeptide, or part of a MASP-3 polypeptide, or DNA encoding a MASP-3 polypeptide, as defined in any of the claims 1-10 to an animal with the aim of producing antibody.

21. An antibody that selectively binds to a MASP-3 polypeptide as defined in any of claims 1 to 10.

22. The antibody according to any of claims 20 and 21, wherein said antibody is a monoclonal antibody or a genetically engineered antibody or an antibody fragment.

23. The antibody according to any of claims 20 to 22, said antibody being coupled to a compound comprising a detectable marker.

24. A compound capable of Inhibiting the complex formation of MBL and MASP-3, wherein said compound comprises a polypeptide as defined in any of claims 1-10.

25. A compound capable of inhibiting the complex formation of MBL and MASP-3, wherein said compound comprises an antibody as defined in any of claims 20 to 23.

26. A compound capable of disrupting the complex formation of MBL and MASP-3, wherein said compound comprises a polypeptide as defined in any of claims 1-10.

27. A compound capable of disrupting the complex formation of MBL and MASP-3, wherein said compound comprising an antibody as defined in any of claims 20 to 23.

28. A compound capable of competitively inhibiting serine protease activity of MASP-3 or a fragment thereof, said compound comprising a polypeptide as defined in any of claims 1-10.

29. A compound capable of competitively inhibiting serine protease activity of MASP-3 or a fragment thereof, said compound comprising an antibody as defined in any of claims 20 to 23.

30. A pharmaceutical composition comprising the polypeptide as defined in any of the claims 1-10, or an antibody as defined in any of the claims 20 to 23, or a compound as defined in any of the claims 24 to 29.

32. The method according to claim 31, in which the specific binding partner is an antibody according to any of the claims 20 to 23.

42. The method according to claim 28, wherein the salt is selected from NaCl, KCl, MgCl₂, CaCl₂, Nal, KCl, MgI₂, CaI₂, from NaBr, KBr, MgBr₂, CaBr₂, Na₂S₂O₃, (NH₄)₂SO₄, and NH₄HCO₃.

44. A method for detecting MASP-3 nucleic acid expression, comprising detecting RNA having a sequence encoding a MASP-3 polypeptide by mixing the sample with a nucleic acid probe that specifically hybridizes under stringent conditions to the nucleic acid as defined in any of claims 11 to 13.

45. A method for treating patients deficient in MASP-3 by administering to the patient the polypeptide as defined in any of claims 1 to 10.

46. A method for treating patients deficient in MASP-3 by administering to the patient nucleic acid as defined in any of claims 11 to 13.

50. The method of claim 49, wherein the compound is as defined by any of the claims 24 to 29.

59. The use of a polypeptide as defined in any of the claims 1-10 for preparation of a pharmaceutical composition.

60. The use according to claim 59, wherein the pharmaceutical composition is capable of being administered parenterally, such as intramusculary, intravenously, or subcutaneously.

61. The use according to claim 59, wherein the pharmaceutical composition is capable of being administered orally.

62. The use according to any of the claim 59 to 61, wherein the pharmaceutical composition is suitable for the treatment of MASP-3 deficiency.

63. The use according to any of the claim 59 to 61, wherein the pharmaceutical composition is suitable for the treatment of immunesystem diseases, or of recoxygenated ischemic tissue.

64. The use of a compound as defined in any of the claims 24 to 29 for preparation of a pharmaceutical composition.

65. The use according to claim 64, wherein the pharmaceutical composition is capable of being administered parenterally, such as intramusculary, intravenously, or subcutaneously.

66. The use according to claim 64, wherein the pharmaceutical composition is capable of being administered orally.

67. The use according to any of the claim 64 to 66, wherein the pharmaceutical composition is suitable for the treatment of aberrant MASP-3 activity.

68. The use according to any of the claim 64 to 66 wherein the pharmaceutical composition is suitable for the treatment of infections, cancer, MBL-deficiency, disorders of the immunesystem and reproductive system.

31. A method for detecting mannan-binding lectin associated serine protease-3 (MASP-3) in a biological sample, said method comprising:

(a) obtaining a biological sample;

(b) contacting said biological sample with a MASP-3 polypeptide specific binding partner that specifically binds MASP-3; and

(c) detecting said complexes, if any, as an indication of the presence of mannin-binding lectin associated serine protease-3 in said sample.

33. The method according to claim 31, wherein the specific binding partner is a mannan-binding lectin (MBL).

34. A method for determing the activity of MASP-3, said method comprising an assay for MASP-3 activity, comprising the steps of

a) applying a sample comprising MBL/MASP-2 complexes to a solid phase obtaining a bound complexes,

b) applying a predetermined amount of MASP-3 to the bound complexes

c) applying at least one complement factor to the complexes,

d) detecting the amount of cleaved complement factors,

e) correlating the amount of cleaved complement factors to the MASP-3 amount, and

f) determining the activity of MASP-3.

35. The method according to claim 34, wherein the solid phase is a mannan coating.

36. The method according to any of the preceding claims 34 to 35, wherein the at least one complement factor is a complement factor cleavable by the MBL/MASP-2 complex.

37. The method according to any of the preceding claims 34 to 36, wherein the at least one complement factor is selected from C3, C4, and C5, preferably C4.

38. The method according to any of the preceding claims 34 to 37, wherein the cleaved complement factor is detected by means of antibodies directed to the complement factor.

39. The method according to any of the preceding claims 34 to 38, wherein activation of the classical complement pathway is inhibited.

40. The method according to claim 39, wherein the activation is inhibited by conducting the assay at high ionic strength.

41. The method according to claim 40, wherein the salt concentration is in the range of from 0.3 M to 10 M, such as from 0.5 M to 5 M, such as from 0.7 M to 2 M, such as from 0.9 M to 2 M, such as about 1.0 M.

43. The method according to any of the claims 34 to 42 for quantitative assay of MASP-3 or MASP-3 activity in biological samples.

47. A method for inhibiting the activity of MASP-3 by administering to the subject a compound that inhibits expression or activity of MASP-3.

48. The method of claim 47 in which the compound is a MASP-3 anti-sense nucleic acid sequence.

49. The method of claim 47 comprising administering a compound that inhibits complexing of MBL and MASP-3.

51. An assay for polymorphisms in the nucleic acid sequence encoding MASP-3.

52. A method of detecting the presence of MASP-3-encoding nucleic acid in a sample, comprising mixing the sample with at least one nucleic acid probe capable of forming a complex with MASP-3-encoding nucleic acid under stringent conditions, and determining whether the probe is bound to sample nucleic acid.

53. A nucleic acid probe capable of forming a complex with MASP-3-encoding nucleic acid under stringent conditions.

54. The nucleic acid probe according to claim 53, being a nucleic acid sequence capable of hybridizing to a nucleic acid sequence identical to SEQ ID NO 4.

55. The nucleic acid probe according to claim 53 to 54, being an anti-sense nucleic acid with respect to a nucleic acid sequence encoding MASP-3.

56. An assay for polymorphisms in the polypeptide sequence comprising MASP-3 or its precursor.

57. A method for diagnosing a disorder associated with aberrant expression of MASP-3, comprising obtaining a biological sample from a patient and measuring MASP-3 expression in said biological sample, wherein increased or decreased MASP-3 expression in said biological sample compared to a control indicates that said patient suffers from a disorder associated with aberrant expression of MASP-3.

58. A method for diagnosing a disorder associated with aberrant activity of MASP-3, comprising obtaining a biological sample from a patient and measuring MASP-3 activity in said biological sample, wherein increased or decreased MASP-3 activity in said biological sample compared to a control indicates that said patient suffers from a disorder associated with aberrant activity of MASP-3.