Protein complexes having Factor VIII:C activity and production thereof

• US 6,060,447 A
• Filed: 05/16/1995
• Issued: 05/09/2000
• Est. Priority Date: 05/19/1987
• Status: Expired due to Term

• Current Assignee: Novartis Vaccines And Diagnostics Incorporated (Novartis Ag)
• Original Assignee: Chiron Corporation (Bayer AG), Novo Nordisk A/S
• Inventors: Burke, Rae Lyn, Chapman, Barbara, Mikkelson, Jan Moller, Rasmussen, Mirella Ezban
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US08/441,935
• Time in Patent Office: 1,820 Days
• Field of Search: 435/69.6, 435/172.3, 435/476, 530/383, 930/100, 514/2, 514/12
• US Class Current: 514/13.7
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/37 Factors VIII
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Recombinant protein complexes having human Factor VIII:C activity are expressed in a eukaryotic host cell by transforming the host cell with first and second expression cassettes encoding a first polypeptide substantially homologous to human Factor VIII:C A domain and a second polypeptide substantially homologous to human Factor VIII:C C domain, respectively. In the present invention, the first polypeptide may be extended having at its C-terminal a human Factor VIII:C B domain N-terminal peptide, a polypeptide spacer of 3-40 amino acids, and a human Factor VIII:C B domain C-terminal peptide. Expression of the second polypeptide is improved by employing an α₁ -antitrypsin signal sequence.

Claims

1. A composition comprising Factor VIII:

C proteins, wherein the Factor VIII;

C proteins consist essentially of a first polypeptide comprising an amino acid sequence of the A domain of human Factor VIII;

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190) or an amino acid sequence that differs therefrom in having not more than 10 number % amino acid substitutions, and a second polypeptide comprising an amino acid sequence of the C domain of human Factor VIII;

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190) or an amino acid sequence that differs therefrom in having not more than 10 number % amino acid substitutions.

2. The composition of claim 1, comprising a complex of the first and second polypeptides.

3. The composition of claim 2, wherein the complex has human Factor VIII:

C coagulant activity.

4. The composition of claim 1, comprising a Ca⁺⁺ -bridged complex of the first and second polypeptides.

5. The composition of claim 4, wherein the complex has human Factor VIII:

C coagulant activity.

6. The composition of claim 2, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-740 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190), and the second polypeptide consists of the amino acid sequence of amino acids 1649-2332 of human Factor VIII;

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

7. The composition of claim 2, wherein the first polypeptide consists of amino acids 1-740 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190) or an amino acid sequence that differs therefrom in having not more than 10 number % amino acid substitutions, and the second polypeptide consists of amino acids 1649-2332 of human Factor VIII;

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190) or an amino acid sequence that differs therefrom in having not more than 10 number % amino acid substitutions.

8. The composition of claim 4, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-740 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190), and the second polypeptide consists of the amino acid sequence of amino acids 1649-2332 of human Factor VIII;

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

9. The composition of claim 4, wherein the first polypeptide consists of amino acids 1-740 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190) or an amino acid sequence that differs therefrom in having not more than 10 number % amino acid substitutions, and the second polypeptide consists of amino acids 1649-2332 of human Factor VIII;

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190) or an amino acid sequence that differs therefrom in having not more than 10 number % amino acid substitutions.

10. The composition of claim 2, wherein the first polypeptide comprises the amino acid sequence of amino acid 1 of the A domain to an amino acid selected from the group consisting of amino acids 741-760 of the B domain as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No. 40190) or an amino acid sequence that differs therefrom in having not more than 10 number % amino acid substitutions, and the second polypeptide comprises the amino acid sequence of amino acids 1649-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

11. The composition of claim 4, wherein the first polypeptide comprises the amino acid sequence of amino acid 1 of the A domain to an amino acid selected from the group consisting of amino acids 741-760 of the B domain as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No. 40190) or an amino acid sequence that differs therefrom in having not more than 10 number % amino acid substitutions, and the second polypeptide comprises the amino acid sequence of amino acids 1649-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

12. The composition of claim 2, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-1102 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

13. The composition of claim 2, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-1315 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

14. The composition of claim 2, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-1405 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

15. The composition of claim 2, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-978 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

16. The composition of claim 2, wherein the first polypeptide consists of the amino acid sequence extending to an amino acid selected from the group consisting of amino acids 741-760 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

17. The composition of claim 4, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-1102 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

18. The composition of claim 4, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-1315 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

19. The composition of claim 4, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-1405 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

20. The composition of claim 4, wherein the first polypeptide consists of the amino acid sequence of amino acids 1-978 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

21. The composition of claim 4, wherein the first polypeptide consists of the amino acid sequence extending to an amino acid selected from the group consisting of amino acids 741-760 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

22. The composition of claim 2, wherein not more than about 5 number % of the amino acids of the amino acid sequence of the first polypeptide are substituted.

23. The composition of claim 4, wherein not more than about 5 number % of the amino acids of the amino acid sequence of the first polypeptide are substituted.

24. The composition of claim 2, wherein the second polypeptide consists of the amino acid sequence from an amino acid selected from the group consisting of amino acids 1570-1649 to an amino acid selected from the group consisting of amino acids 2300-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

25. The composition of claim 24, wherein the second polypeptide consists of the amino acid sequence from an amino acid selected from the group consisting of amino acids 1600-1649 to an amino acid selected from the group consisting of amino acids 2310-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

26. The composition of claim 25, wherein the second polypeptide consists of the amino acid sequence from an amino acid selected from the group consisting of amino acids 1625-1649 to an amino acid selected from the group consisting of amino acids 2325-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

27. The composition of claim 26, wherein the second polypeptide consists of the amino acid sequence from an amino acid selected from the group consisting of amino acids 1640-1649 to an amino acid selected from the group consisting of amino acids 2325-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

28. The composition of claim 4, wherein the second polypeptide consists of the amino acid sequence from an amino acid selected from the group consisting of amino acids 1570-1649 to an amino acid selected from the group consisting of amino acids 2300-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

29. The composition of claim 28, wherein the second polypeptide consists of the amino acid sequence from an amino acid selected from the group consisting of amino acids 1600-1649 to an amino acid selected from the group consisting of amino acids 2310-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

30. The composition of claim 29, wherein the second polypeptide consists of the amino acid sequence from an amino acid selected from the group consisting of amino acids 1625-1649 to an amino acid selected from the group consisting of amino acids 2325-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

31. The composition of claim 30, wherein the second polypeptide consists of the amino acid sequence from an amino acid selected from the group consisting of amino acids 1640-1649 to an amino acid selected from the group consisting of amino acids 2325-2332 of human Factor VIII:

C as encoded by the polynucleotide present in plasmid pSVF8-200 (ATCC No.

     40190).

32. A pharmaceutical composition comprising the composition of claim 1 and a physiologically acceptable carrier.

33. A pharmaceutical composition comprising the composition of claim 4 and a physiologically acceptable carrier.

34. A method for treating an individual requiring Factor VIII:

C activity comprising administering to the individual a sufficient amount of the protein complex of claim 32 to enhance blood clotting activity in the individual.

35. A method for treating an individual requiring Factor VIII:

C activity comprising administering to the individual a sufficient amount of the protein complex of claim 33 to enhance blood clotting activity in the individual.