Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
MODIFIED VITAMIN K-DEPENDENT POLYPEPTIDES
Document Type and Number:
WIPO Patent Application WO/2000/066753
Kind Code:
A2
Abstract:
The invention provides vitamin K-dependent polypeptides with enhanced membrane binding affinity. These polypeptides can be used to modulate clot formation in mammals. Methods of modulating clot formation in mammals are also described.

Inventors:
NELSESTUEN GARY L (US)
Application Number:
PCT/US2000/011416
Publication Date:
November 09, 2000
Filing Date:
April 28, 2000
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
UNIV MINNESOTA (US)
NELSESTUEN GARY L (US)
International Classes:
A61K38/00; A61K38/43; A61P7/02; A61P7/04; C07K14/745; C12N5/10; C12N9/64; C12N15/12; C12N15/09; C12N15/55; C12N15/57; C12P21/02; G01N33/15; G01N33/50; (IPC1-7): C12N15/57; C12N15/12; C12N9/64; C07K14/745; A61K38/36; A61K38/48; C12N5/10; C12Q1/37
Domestic Patent References:
WO1999020767A11999-04-29
Other References:
SHEN L ET AL: "Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: studies related to proline-10" BIOCHEMISTRY,US,AMERICAN CHEMICAL SOCIETY. EASTON, PA, vol. 36, no. 51, 23 December 1997 (1997-12-23), pages 16025-16031, XP002092844 ISSN: 0006-2960
SHEN LEI ET AL: "Enhancement of human protein C function by site-directed mutagenesis of the gamma-carboxyglutamic acid domain." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 47, 20 November 1998 (1998-11-20), pages 31086-31091, XP002149742 ISSN: 0021-9258
MCDONALD J F ET AL: "Comparison of naturally occurring vitamin K - dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site" BIOCHEMISTRY,US,AMERICAN CHEMICAL SOCIETY. EASTON, PA, vol. 36, no. 17, 29 April 1997 (1997-04-29), pages 5120-5127, XP002092840 ISSN: 0006-2960
See also references of EP 1177304A2
Attorney, Agent or Firm:
Ellinger, Mark S. (P.A. 60 South Sixth Street Suite 3300 Minneapolis, MN, US)
Download PDF:
Claims:
What is claimed is:
1. A vitamin Kdependent polypeptide comprising a modified GLA domain that enhances membrane binding affinity and activity of said polypeptide relative to a corresponding native vitamin Kdependent polypeptide, said modified GLA domain comprising at least one amino acid substitution, wherein said polypeptide is one that inhibits clot formation.
2. The polypeptide of claim 1, wherein said amino acid substitution is at amino acid 5,9,11,12,29,33,34,35, or 36.
3. The polypeptide of claim 1, wherein said amino acid substitution is at amino acid 5,9,35, or 36.
4. The polypeptide of claim 1, wherein said amino acid substitution is at amino acid 11 or 12.
5. The polypeptide of claim 1, wherein said amino acid substitution is at amino acid 29 or 33.
6. The polypeptide of claim 1, wherein said amino acid substitution is at amino acid 34, 35, or 36.
7. The polypeptide of claim 1, wherein said polypeptide comprises Protein C or Activated Protein C.
8. The polypeptide of claim 7, wherein said amino acid substitution comprises a glycine residue at amino acid 12.
9. The polypeptide of claim 7, wherein said amino acid substitution comprises a glutamic acid residue at amino acid 33.
10. The polypeptide of claim 8, wherein said amino acid substitution further comprises a glutamic acid residue at amino acid 33.
11. The polypeptide of claim 9 or claim 10, wherein said amino acid substitution further comprises an aspartic acid or glutamic acid residue at amino acid 35.
12. The polypeptide of claim 9 or claim 10, wherein said amino acid substitution further comprises or a glutamic acid residue at amino acid 36.
13. The polypeptide of claim 9 or claim 10, wherein said amino acid substitution further comprises a glutamine or a glutamic acid residue at amino acid 11.
14. The polypeptide of claim 9 or claim 10, wherein said amino acid substitution further comprises a phenylalanine residue at amino acid 29.
15. The polypeptide of claim 9, claim 10, or claim 11, wherein said amino acid substitution further comprises an aspartic acid, glutamic acid, phenylalanine, leucine, or isoleucine residue at amino acid 34.
16. The polypeptide of claim 1, wherein said polypeptide comprises activesite modified Factor VIIa.
17. The polypeptide of claim 16, wherein said polypeptide comprises a glutamic acid at amino acid 33.
18. The polypeptide of claim 16, wherein said amino acid substitution comprises a glutamine residue at amino acid 11 and a glutamic acid residue at amino acid 33.
19. The polypeptide of claim 17 or claim 18, wherein said amino acid substitution further comprises a phenylalanine, leucine, or isoleucine residue at amino acid 34.
20. The polypeptide of claim 17, claim 18, or claim 19, wherein said amino acid substitution further comprises an aspartic acid or a glutamic acid residue at amino acid 35.
21. The polypeptide of claim 1, wherein said polypeptide is Protein S.
22. The polypeptide of claim 21, wherein said amino acid substitution comprises an isoleucine, leucine, valine, or phenylalanine residue at amino acid 9.
23. The polypeptide of claim 22, wherein said amino acid substitution further comprises a phenylalanine, leucine, isoleucine, aspartic acid, or glutamic acid residue at amino acid 34 or an aspartic acid or glutamic acid residue at amino acid 35.
24. The polypeptide of claim 21, wherein said amino acid substitution comprises a phenylalanine residue at amino acid 5.
25. The polypeptide of claim 21, wherein said polypeptide further comprises a substitution in the thrombinsensitive loop.
26. The polypeptide of claim 25, wherein said substitution in the thrombin sensitive loop is at amino acid 49,60, or 70.
27. The polypeptide of claim 1, wherein said polypeptide is activesite modified Factor IXa.
28. The polypeptide of claim 27, wherein said amino acid substitution comprises a phenylalanine at amino acid 29.
29. The polypeptide of claim 27, wherein said amino acid substitution comprises a phenylalanine residue at amino acid 5.
30. The polypeptide of claim 27, wherein said amino acid substitution comprises a phenylalanine, leucine, or isoleucine at amino acid 34 and an aspartic acid or glutamic acid residue at amino acid 35.
31. The polypeptide of claim 1, wherein said vitamin Kdependent polypeptide further comprises an inactivated cleavage site.
32. The polypeptide of claim 31, wherein said polypeptide comprises factor VII.
33. The polypeptide of claim 32, wherein said inactivated cleavage site comprises a substitution of an alanine residue at amino acid 152.
34. The polypeptide of claim 1, wherein said polypeptide is active site modified factor Xa.
35. The polypeptide of claim 33, wherein said amino acid substitution comprises a glutamine at amino acid 11.
36. The polypeptide of claim 34, wherein said amino acid substitution comprises a phenylalanine, leucine, or isoleucine at amino acid 34 and an aspartic acid or glutamic acid residue at amino acid 35.
37. The polypeptide of claim 1, wherein said polypeptide is protein Z.
38. The polypeptide of claim 37, wherein said amino acid substitution comprises a phenylalanine, leucine, or isoleucine residue at amino acid 34, or an aspartic acid or glutamic acid residue at amino acid 35.
39. A vitamin Kdependent polypeptide comprising a modified GLA domain that enhances membrane binding affinity and activity of said polypeptide relative to a corresponding native vitamin Kdependent polypeptide, said modified GLA domain comprising at least one amino acid insertion at amino acid 4.
40. The polypeptide of claim 39, wherein said polypeptide is selected from the group consisting of factor VII or factor VIIa, protein C or activated protein C, factor X or factor Xa, and protein S.
41. The polypeptide of claim 39, wherein said polypeptide is factor VII, factor VIIa, protein C, or activated protein C.
42. The polypeptide of claim 41, wherein said amino acid insertion comprises a tyrosine residue.
43. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a vitamin Kdependent polypeptide, wherein said vitamin Kdependent polypeptide comprises a modified GLA domain that enhances membrane binding affinity and activity of said polypeptide relative to a corresponding native vitamin Kdependent polypeptide, said modified GLA domain comprising at least one amino acid substitution, and wherein said vitamin K dependent polypeptide inhibits clot formation.
44. The pharmaceutical composition of claim 43, wherein said polypeptide is Protein C or Activated Protein C.
45. The pharmaceutical composition of claim 44, wherein said amino acid substitution comprises a glycine at amino acid 12.
46. The pharmaceutical composition of claim 45, wherein said amino acid substitution further comprises a glutamic acid residue at amino acid 33.
47. The pharmaceutical composition of claim 45, wherein said amino acid substitution further comprises a phenylalanine, leucine, or isoleucine residue at amino acid 34, and an aspartic acid or glutamic acid residue at amino acids 35 or 36.
48. The pharmaceutical composition of claim 43, wherein said polypeptide is activesite modified Factor VIIa.
49. The pharmaceutical composition of claim 48, wherein said amino acid substitution comprises a glutamine residue at amino acid 11, a glutamic acid residue at amino acid 33, a phenylalanine, leucine, or isoleucine residue at amino acid 34, and an aspartic acid or glutamic acid residue at amino acid 35.
50. The pharmaceutical composition of claim 43, wherein said polypeptide is Protein S or activesite modified Factor IXa.
51. The pharmaceutical composition of claim 43, wherein said composition further comprises an anticoagulant agent.
52. A mammalian host cell comprising a vitamin Kdependent polypeptide, said vitamin Kdependent comprising a modified GLA domain that enhances membrane binding affinity and activity of said polypeptide relative to a corresponding native vitamin Kdependent polypeptide, said modified GLA domain comprising at least one amino acid substitution, wherein said polypeptide is one that inhibits clot formation.
53. A method of decreasing clot formation in a mammal comprising administering an amount of a vitamin Kdependent polypeptide effective to decrease clot formation in said mammal, wherein said vitamin Kdependent polypeptide comprises a modified GLA domain that enhances membrane binding affinity and activity of said polypeptide relative to a corresponding native vitamin Kdependent polypeptide, said modified GLA domain comprising at least one amino acid substitution.
54. The method of claim 53, wherein said polypeptide is Protein C or Activated protein C, activesite modified Factor VIIa, activesite modified Factor IXa, or Protein S.
55. A method for identifying a vitamin Kdependent polypeptide having enhanced membrane binding affinity and activity comprising: (a) modifying the GLA domain of said vitamin Kdependent polypeptide, wherein said modifying comprises substituting at least one amino acid in said GLA domain; b) monitoring membrane binding affinity and activity of said vitamin K dependent polypeptide having said modified GLA domain; and (c) identifying said modified vitamin Kdependent polypeptide as having enhanced membrane binding affinity and activity if membrane binding affinity and activity of said modified vitamin Kdependent polypeptide is enhanced relative to a corresponding native vitamin Kdependent polypeptide.
56. The method of claim 55, wherein said amino acid substitution is at amino acid 5,9,11,12,29,33,34,35, or 36.
57. The method of claim 55, wherein said modified vitamin Kdependent polypeptide increases clot formation.
58. The method of claim 55, wherein said modified vitamin Kdependent polypeptide inhibits clot formation.
59. A Factor VII or Factor IX polypeptide comprising a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native Factor VII or Factor IX polypeptide, said modified GLA domain comprising at least one amino acid substitution at residue 11,29,34, or 35.
60. The polypeptide of claim 59, wherein said polypeptide comprises Factor VII or Factor VIIa.
61. The polypeptide of claim 60, wherein a glutamine, a glutamic acid, an aspartic acid, or an asparagine residue is substituted at amino acid 11.
62. The polypeptide of claim 60, wherein a glutamine residue is substituted at amino acid 11.
63. The polypeptide of claim 59 or claim 60, wherein a glutamic acid or a phenylalanine residue is substituted at amino acid 29.
64. The polypeptide of claim 60, wherein said modified domain further comprises an amino acid substitution at amino acid 33.
65. The polypeptide of claim 64, wherein a glutamic acid residue is substituted at amino acid 33.
66. The polypeptide of claim 61 or claim 63, wherein said modified GLA domain further comprises a substitution of a glutamic acid residue at amino acid 33.
67. The polypeptide of claim 60, wherein said modified GLA domain further comprises at least one hydrophobic residue at residue 34 or 35.
68. The polypeptide of claim 60, wherein said modified GLA domain further comprises a phenylalanine, leucine, or isoleucine residue at amino acid 34 and an aspartic acid or glutamic acid residue at amino acid 35.
69. The polypeptide of claim 59, wherein said polypeptide comprises Factor IX or Factor IXa.
70. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of a Factor VII or Factor IX polypeptide effective to increase clot formation in a mammal, wherein said Factor VII or Factor IX polypeptide comprises a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native Factor VII or Factor IX polypeptide, said modified GLA domain comprising at least one amino acid substitution at residue 11,29,34, or 35.
71. The pharmaceutical composition of claim 70, wherein said polypeptide further comprises an amino acid substitution at amino acid 33.
72. The pharmaceutical composition of claim 70, wherein said pharmaceutical composition further comprises soluble tissue factor.
73. A method of increasing clot formation in a mammal comprising administering an amount of a Factor VII or Factor IX polypeptide effective to increase clot formation in said mammal, wherein said Factor VII or Factor IX polypeptide comprises a modified GLA domain that enhances membrane binding affinity of said polypeptide relative to a corresponding native Factor VII or Factor IX polypeptide, said modified GLA domain comprising at least one amino acid substitution at residue 11,29,34, or 35.
74. A method for treating a bleeding disorder in a patient, said method comprising administering the pharmaceutical composition of claim 73 to said patient.
75. An isolated nucleic acid molecule, said molecule comprising a nucleic acid sequence encoding the polypeptide of claim 1 or claim 59.
Description:
C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category ° Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X SHEN LEI ET AL:"Enhancement of human 1,2,4, protein C function by site-directed 7-10,13, mutagenesis of the gamma-carboxyglutamic 43,44, aciddomain."46, JOURNAL OF BIOLOGICAL CHEMISTRY, 51-58,75 vol. 273, no. 47, 20 November 1998 (1998-11-20), pages 31086-31091,XP002149742 ISSN:0021-9258 Y the whole document 2,3 Y MCDONALD J F ET AL :"Comparison of 2,3 naturally occurring vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site" BIOCHEMISTRY, US, AMERICAN CHEMICAL SOCIETY. EASTON, PA, vol. 36, no. 17, 29 April 1997 (1997-04-29), pages 5120-5127,XP002092840 ISSN:0006-2960 the whole document 2 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This International Search Report has not been established in respect of certain claims under Article 17 (2) (a) for the following reasons: 1. X Claims Nos. : because they relate to subject matter not required to be searched by this Authority, namely: Although claims 53 and 54 are directed to a method of treatment of the human/animal body, the search has been carried out and based on the alleged effects of the compound/composition. ry1 2. Claims Nos. : because they relate to parts of the International Application that do not comply with the prescribed requirements to such an extent that no meaningful International Search can be carried out, specifically: 3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4 (a). Box 11 Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows: see additional sheet 1. As all required additional search fees were timely paid by the applicant, this International Search Report covers all searchable claims. 2. as ait searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this International Search Report covers only those claims for which fees were paid, specifically claims Nos.: 4. No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Claims 7-15 compeletely, 1-6,31,39-43,51-58,75 partially. Remark on Protest ! The additional search fees were accompanied by the applicant's protest. No protest accompanied the payment of additional search fees. u FURTHER INFORMATION CONTINUED FROM PCT/ISA/210 This International Searching Authority found multiple (groups of) inventions in this international application, as follows : 1. Claims: 7-15 44-47 completely, 1-6 31 39-43 51-58 75 partially Protein C with modified GLA domain, its use and nucleic acids encoding it 2. Claims: 16-20 32 33 48 49 60-62 64 65 67 68 completely 1-6 31 39-43 51-59 63 66 70-75 partially Factor VII with modified GLA domain, its use and nucleic acids encoding it 3. Claims: 21-26 completely 1-6 31 39 40 43 50-58 75 partially Protein S with modified GLA domain, its use and nucleic acids encoding it 4. Claims: 27-30 69 completely 1-6 31 39 43 50-59 63 66 70-75 partially Factor IX with modified GLA domain, its use and nucleic acids encoding it 5. Claims : 34-36 1-6 31 39 40 43 51-53 55-58 75 partially Factor X with modified GLA domain, its use and nucleic acids encoding it 6. Claims: 37 38 completely 1-6 31 39 43 51-53 55-58 75 partially Protein Z with modified GLA domain, its use and nucleic acids encoding it Patent document Publication Patent family Publication cited in search report date member (s) date WO 9920767 A 29-04-1999 US 6017882 A 25-01-2000 AU 2702499 A 10-05-1999 BR 9814611 A 03-10-2000 NO 20002025 A 19-06-2000 ZA 9809597 A 23-04-1999