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1. WO1996039503 - C5a RECEPTOR ANTAGONISTS HAVING SUBSTANTIALLY NO AGONIST ACTIVITY AND METHODS FOR PREPARATION

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[ EN ]
Claims:

1. A polypeptide derivative of human C5a, wherein said derivative is a C5a receptor antagonist that exhibits substantially no agonist activity, said derivative having a glycine residue as the N-terminus.

2. The derivative according to claim 1 , wherein said glycine residue is in the form of an adduct.

3. The derivative according to claim 1 , wherein said glycine residue is a substituent for the N-terminal amino acid residue Thr of human C5a.

4. The derivative according to claim 1 , wherein said derivative comprises a C-terminal region which differs from the corresponding C-terminal region of human C5a, in that it has a cysteine residue, and is truncated at its C-terminus by at least two amino acid residues.

5. The derivative according to claim 4, having a cysteine residue as the C-terminus.

6. The derivative according to claim 4, wherein said cysteine residue is in the form of an adduct.

7. The derivative according to claim 4, which is from 64 to 72 amino acids in length.

8. The derivative according to claim 7, which is from 68 to 72 amino acids in length.

9. The dervative according to claim 8, which is from 70 to 72 amino acids in length.

10. The derivative according to claim 9, which is 71 amino acids in length.

11. The derivative of claim 10, which is C5a (1 -71 , Thr1 Gly, Cys27Ser, Gln71 Cys).

12. The derivative of claim 10, which is C5a (1-71 , Thrl Gly, Cys27Ser, His67Phe,
Gln71Cys).

13. A dimer, comprising first and second polypeptide derivative of human C5a, wherein each of said derivatives is a C5a receptor antagonist that exhibits substantially no agonist activity, and has a C-terminal cysteine residue, and wherein the cysteine residues of said first and second derivatives are linked together via a disulfide linkage, and wherein said first and second polypeptide derivative of human C5a may be the same or different, and further wherein at least one of said first and second polypeptide derivatives of human C5a is a polypeptide derivative of human C5a as defined in claim 1.

14. The dimer according to claim 13, wherein the C-terminal region of each of said first and said second derivatives differs from the corresponding C-terminal region of human C5a in that-it is truncated by at least two amino acid Tesidues.

15. The dimer of claim 14, wherein each of said first and second derivatives is the human C5a derivative C5a (1-71 , ThrlGly, Cys27Ser, Gln71 Cys).

16. A fusion protein, consisting of, in the order of N-terminus to C-terminus, a fusion partner, a cleavable linker and, fused thereto, a polypeptide derivative of human C5a, wherein said polypeptide derivative is a C5a receptor antagonist that exhibits substantially no agonist activity.

17. The fusion protein according to claim 16, wherein said derivative comprises a C-terminal region which differs from the corresponding C-terminal region of human C5a, in that it has a cysteine residue, and is truncated at its C-terminus by at least two amino acid residues.

18. The fusion protein according to claim 17, wherein said derivative has a cysteine residue as the C-terminus.

19. The fusion protein according to claim 17, wherein said derivative has a glycine residue as the N-terminus.

20. The fusion protein according to claim 16, wherein said fusion partner is a polypeptide capable of directing the formation of inclusion bodies in a cell.

21. The fusion protein according to claim 20, wherein said cell is an E. coli cell.

22. The fusion protein according to claim 20, wherein said fusion partner together with said cleavable linker is a polypeptide about the amino acid length of said polypeptide derivative of human C5a.

23. The fusion protein according to claim 22, wherein said fusion partner comprises an N-terminal fragment of human IL-1 β or a mutant thereof.

24. The fusion protein according to claim 23, wherein said fusion partner consists of amino acid residues 1 to 47 of human IL-1 β or a mutant thereof and amino acid residues 52 to 57 of human IL-1 RA.

25. The fusion protein according to claim 23, wherein said fusion partner consists of amino acid residues 1 to 47 of human IL-1β or a mutant thereof and amino acid residues 52 to 54 of human IL-1 RA.

26. The fusion protein acccording to claim 16, wherein said linker comprises a
hydroxylamine cleavage site.

27. The fusion protein according to claim 26, wherein said linker comprises, in the order of N-terminus to C-terminus, an enterokinase protease cleavage site and a hydroxylamine cleavage site.

28. The fusion protein according to claim 16, consisting of, in the order of N-terminus to C-terminus, amino acid residues 1 to 47 of human IL-1 β or a mutant thereof , amino acid residues 52 to 54 of human IL-1 RA, a cleavable linker comprising the amino acid sequence -Val-Asp-Asp-Asp-Asp-Lys-Asn-Gly-, and a polypeptide derivative as defined in claim 1 , wherein the C-terminal residue Gly of said linker is a substituent for the N-terminal amino acid residue Thr of human C5a in said polypeptide derivative.

29. The fusion protein according to claim 28, wherein said polypeptide derivative is as defined in claim 7.

30. The fusion protein according to claim 29, wherein said polypeptide derivative is as defined in claim 11.

31. The fusion protein according to claim 29, wherein said polypeptide derivative is as defined in claim 12.

32. A DNA molecule encoding a polypetide derivative of human C5a as defined in claim 1.

33. The DNA molecule according to claim 32, wherein said derivative is as defined in claim 7.

34. The DNA molecule according to claim 33, wherein said derivative is as defined in claim 11.

35. The DNA molecule according to claim 33, wherein said derivative is as defined in claim 12.

36. A DNA molecule encoding a fusion protein as defined in claim 16.

37. The DNA molecule according to claim 36, wherein said fusion protein is as defined in claim 19.

38. The DNA molecule according to claim 37, wherein said fusion protein is as defined in claim 24.

39. The DNA molecule according to claim 37, wherein said fusion protein is as defined in claim 25.

40. The DNA molecule according to claim 37, wherein said fusion protein is as defined in claim 28.

41. A recombinant DNA molecule, comprising a promoter capable of functioning in a given host operably linked to a DNA molecule as defined in claim 32.

42. A recombinant DNA molecule, comprising a promoter capable of functioning in a given host operably linked to a DNA molecule as defined in claim 36.

43. A recombinant plasmid compatible with a given host, comprising a recombinant DNA molecule according to claim 32.

44. A recombinant plasmid compatible with a given host, comprising a recombinant DNA molecule according to claim 36.

45. A recombinant vector compatible with a given host, comprising a recombinant DNA molecule according to claim 32.

46. A recombinant vector compatible with a given host, comprising a recombinant DNA molecule according to claim 36.

47. A recombinant host, stably transformed with a recombinant DNA molecule according to claim 32.

48. The recombinant host according to claim 47, selected from the group consisting of bacterial, yeast, fungal, insect, mammalian and plant cells.

49. The recombinant host according to claim 48, which is E. coli.

50. A recombinant host, stably transformed with a recombinant DNA molecule according to claim 36.

51. The recombinant host according to claim 50, selected from the group consisting of bacterial, yeast, fungal, insect, mammalian and plant cells.

52. The recombinant host according to claim 51 , which is E. coli.

53. A method of preparing a biologically active polypeptide derivative of human C5a as defined in claim 1 , comprising the steps of:
(1) culturing E. coli cells stably transformed with a DNA molecule encoding said polypeptide derivative under conditions suitable to cause expression of the DNA molecule;
(2) contacting the thus-cultured cells with a denaturing and solubilizing agent to produce said polypeptide derivative in denatured form; and
(3) mixing the thus-denatured polypeptide derivative with a solution containing a reducing agent and an oxidizing agent in a molar ratio of the reducing agent to the oxidizing agent by weight of at least about 100:1 under suitable conditions to produce the polypeptide derivative in biologically active form.

54. The method according to claim 53, wherein said DNA molecule encodes the polypetide derivative in the form of a fusion protein.

55. The method according to claim 54, further comprising the step of cleaving the thus-expressed fusion protein prior to said step of mixing.

56. The method according to claim 54, wherein said DNA molecule encodes a polypeptide derivative of human C5a, which is from 64 to 72 amino acid residues in length.

57. The method according to claim 56, wherein the polypeptide derivative is C5a (1 -71 , ThrlGly, Cys27Ser, Gln71Cys).

58. A method according to claim 58, wherein the polypeptide derivative is C5a (1-71 , ThrlGly, Cys27Ser, His67Phe, Gln71Cys).

59. A method of preparing a biologically active polypeptide derivative of human C5a, wherein said derivative is a C5a receptor antagonist that exhibits substantially no agonist activity, comprising the steps of:
(1) culturing host cells stably transformed with a recombinant DNA molecule encoding said polypeptide derivative in the form of a fusion protein wherein said culturing is conducted under conditions suitable to cause expression of said fusion protein in the form of inclusion bodies;
(2) isolating the inclusion bodies containing the fusion protein from the thus-cultured host cells;
(3) contacting the thus-isolated inclusion bodies with a denaturing and solubilizing agent to produce the fusion protein in denatured form;
(4) cleaving the thus-isolated fusion protein so that the polypeptide derivative of human C5a can be obtained; and
(5) mixing the thus-cleaved fusion protein with a solution containing a reducing agent and an oxidizing agent in a molar ratio of the reducing agent to the oxidizing agent by weight of at least about 100:1 under suitable conditions to produce the polypeptide derivative in biologically active form;
wherein steps 3 and 4 are carried out simultaneously or consecutively.

60. The method according to claim 59, wherein said mixing is conducted at a pH of from about 6.5 to about 7.5.

61. The method according to claim 59, wherein said mixing is conducted for a period of time from about 1/2 hour to about 4 hours.

62. The method according to claim 59, wherein the redox couple is reduced
glutathione/oxidized glutathione.

63. The method according to claim 59, wherein said host cells are E. coli cells.

64. The method according to claim 59, wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 16.

65. The method according to claim 64, wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 19.

66. The method according to claim 65, wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 22.

67. The method according to claim 66, wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 23.

68. The method according to claim 67, wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 24.

69. The method according to claim 67, wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 25.

70. The method according to claim 59, wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 28.

71. The method according to claim 70, wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 29.

72. The method according to claim 71 , wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 30.

73. The method according to claim 71 , wherein said recombinant DNA molecule encodes a fusion protein as defined in claim 31.

74. An antibody specific to the polypeptide derivative as defined in claim 1 or to a domer as defined in claim 13, wherein said antibody exhibits substantially no cross-reactivity with human C5a.

75. An antibody according claim 74, wherein said antibody is polyclonal.

76. An antibody according to claim 74, wherein said antibody is monoclonal.

77. An antibody according to claim 74, specific to the derivative as defined in claim 11.

78. An antibody according to claim 74, specific to a dimer as defined in claim 14.

79. An antibody according to claim 78, specific to the dimer as defined in claim 15.

80. A pharmaceutical composition useful in the treatment of a C5a-mediated disease or inflammatory condition in a mammal inclusive man, comprising a therapeutically effective amount of a polypeptide derivative of human C5a as defined in claim 1 or of a dimer as defined in claim 13, and optionally a pharmaceutically acceptable carrier.

81. A pharmaceutical composition according to claim 80, wherein said derivative is as defined in claim 11 , or said dimer is as defined in claim 15.

82. A pharmaceutical composition useful in modulating the in vivo activity of a polypeptide derivative of human C5a as defined in claim 1 , comprising
an antibody as defined in claim 74 in an amount effective to modulate the activity of the derivative, and optionally a pharmaceutically acceptable carrier.

83. The pharmaceutical composition according to claim 82, wherein the amount of said antibody is effective to substantially neutralize the in vivo activity of said derivative.

84. A method of treating a C5a-mediated disease or inflammatory condition in a mammal, comprising the step of administering a pharmaceutical composition according to claim 80 to a mammal inclusive man in need thereof.

85. A method of reducing C5a-mediated inflammation in a mammal inclusive man, comprising the step of administering a pharmaceutical composition according to claim 80 to said mammal at a time relative to a complement activation-causing or aggravating event sufficient to reduce the inflammation.

86. A method of modulating the activity of a polypeptide derivative as defined in claim 1 or of a dimer as defined in claim 13, in a subject in need thereof, comprising the step of: administering to the subject a pharmaceutical composition according to claim 82.

87. A method of neutralizing the activity of a polypeptide derivative as defined in claim 1 or of a dimer as defined in claim 13 in a subject in need thereof, comprising the step of: administering to the subject a pharmaceutical composition according to claim 83.

88. A qualitative or quantitative assay for the determination of a polypeptide derivative as defined in claim 1 in a subject, comprising the steps of:
(1 ) obtaining a tissue or a fluid sample from the subject, and
(2) contacting the sample with an antibody according to claim 74 under conditions sufficient to allow the detectable formation of an immunocomplex between the antibody and the derivative, wherein the formation of the immunocomplex is indicative of the presence of the derivative in the subject.

89. An assay according to claim 88, further comprising the step of quantifying the derivative in the subject.

90. A polypeptide derivative according to claim 1 or a dimer according to claim 13 for use in the therapeutic treatment of a mammal inclusive man.

91. Use of a polypeptide derivative according to claim 1 or of a dimer according to claim 13 for the preparation of a pharmaceutical composition for treatment of a C5a-mediated disease or inflammatory condition in a mammal inclusive man.