Production and use of multimeric hemoglobins

• US 5,599,907 A
• Filed: 05/09/1994
• Issued: 02/04/1997
• Est. Priority Date: 05/10/1989
• Status: Expired due to Term

• Current Assignee: Baxter Biotech Technology SARL (Baxter International Inc.)
• Original Assignee: Somatogen, Inc. (Baxter International Inc.)
• Inventors: Anderson, David C., Mathews, Antony J., Stetler, Gary L.
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): LEITH, NANCY JILL
• Application Number:
  US08/240,712
• Time in Patent Office: 1,002 Days
• Field of Search: 536/23.4, 536/23.5, 435/69.1, 435/69.6, 435/69.7, 435/71.1, 530/385, 530/829, 514/832
• US Class Current: 530/385
• 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 47/6445 Haemoglobin
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/92 containing an intein ("protein splicing")domain
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: Y10S 530/829 Blood

Abstract

Multimeric hemoglobin-like proteins are obtained by crosslinking cysteines of the component tetramers, or by genetically fusing globin-like domains of one tetramer with those of another, by means of an interdomain spacer sequence. Artificial cysteines are introduced selectively in a single globin-like domain per tetramer to control polymerization.

Claims

1. A non-naturally occuring pseudotetrameric hemoglobin-like protein which is a pseudotetramer comprising four globin-like domains, said pseudotetramer comprising a pseudodimeric polypeptide having two substantially homologous globin-like domains, one of which is mutated to provide an asymmetric crosslinkable cysteine residue, the corresponding residue in the other globin-like domain of said pseudodimeric polypeptide being an amino acid other than cysteine, said pseudotetramer further comprising at least one other globin-like domain-bearing polypeptide.

2. A non-naturally occuring multimeric hemoglobin-like protein composed of two or more pseudotetrameric hemoglobin-like proteins according to claim 1, the asymmetric crosslinkable cysteine residue of a first pseudotetramer being crosslinked to the asymmetric crosslinkable cysteine residue of a second pseudotetramer.

3. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 2, wherein each of the globin-like domains is a mammalian globin-like domain.

4. The protein of claim 2 in which the protein is composed of two pseudotetramers.

5. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 4, wherein each of the globin-like domains is a mammalian globin-like domain.

6. The protein of claim 2 wherein the crosslink comprises a disulfide bond.

7. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 6, wherein each of the globin-like domains is a mammalian globin-like domain.

8. The protein of claim 2 wherein the crosslink is formed by reaction of the pseudotetramers with a multi-functional, thiol-specific crosslinking reagent.

9. The protein of claim 8 wherein the crosslinking reagent forms a crosslink which is substantially less reducible by reducing agents endogenous to plasma than would be a disulfide bond between the same two cysteine residues.

10. The protein of claim 8 wherein the crosslink is a thioether or a thio-maleiimide crosslink.

11. The protein of claim 8 in which the crosslinking reagent is a polyethylene glycol derivative with thiol-specific reactive moieties.

12. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 8, wherein each of the globin-like domains is a mammalian globin-like domain.

13. The protein of claim 8, in which the protein is composed of more than two pseudotetramers.

14. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 13, wherein each of the globin-like domains is a mammalian globin-like domain.

15. The protein of claim 8 in which the crosslinking agent comprises a peptide moiety.

16. The protein of claim 15 in which the crosslinking agent comprises one or more D-amino acids.

17. The protein of claim 15 in which the peptide moiety comprises a stable, extended, secondary structure.

18. The protein of claim 17 in which the peptide moiety comprises a polyproline helix.

19. The protein of claim 17 in which the crosslinking agent comprises a two-stranded coiled coil.

20. The protein of claim 19 in which the two strands of the coiled coil are disulfide cross-linked.

21. The protein of claim 17 in which the crosslinking agent comprises a 4-helical or a 4-stranded coiled coil.

22. The protein of claim 17 in which the peptide moiety comprises a branched chain.

23. The protein of claim 8 wherein the crosslink comprises a negatively charged moiety, whereby the isoelectric point of the protein is reduced.

24. The protein of claim 23 wherein the negatively charged moiety comprises a plurality of Asp and/or Glu residues.

25. The protein of claim 1 wherein the cysteine residue lies in an alpha globin-like domain.

26. The protein of claim 1 wherein the cysteine residue lies in a beta globin-like domain.

27. The protein of claim 1, wherein the hemoglobin-like protein is mutated, relative to human hemoglobin, to inhibit haptoglobin binding.

28. The protein of claim 27 wherein said crosslinking inhibits haptoglobin binding.

29. The protein of claim 1 wherein the globin-like domains of the pseudodimeric polypeptide are joined by a linker moiety consisting essentially of one or more glycines.

30. The protein of claim 29 wherein the domains are alpha globin-like domains and the linker moiety is 1-3 glycines.

31. The protein of claim 29 wherein the domains are beta globin-like domains and the linker moiety is 2-9 glycines.

32. The protein of claim 1, which protein has a P₅₀ at least 10% greater than does human hemoglobin Ao under the same conditions.

33. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 32, wherein each of the globin-like domains is a mammalian globin-like domain.

34. The protein of claim 1, which protein has a P₅₀ at least 10% lower than does human hemoglobin Ao under the same conditions.

35. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 34, wherein each of the globin-like domains is a mammalian globin-like domain.

36. The protein of claim 1, wherein said protein has a P₅₀ of 24-32 torr.

37. The protein of claim 1, wherein said protein has substantially longer intravascular retention than normal hemoglobin free in plasma.

38. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 37, wherein each of the globin-like domains is a mammalian globin-like domain.

39. The protein of claim 1 wherein each of the globin-like domains is a vertebrate globin-like domain.

40. The protein of claim 1 wherein each of the globin-like domains is a mammalian globin-like domain.

41. The protein of claim 1 wherein each of the globin-like domains is a human globin-like domain.

42. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 1, wherein each of the globin-like domains is a vertebrate globin-like domain.

43. The method of claim 42 wherein each of the globin-like domains is a human globin-like domain.

44. The method of claim 42 in which the globin-like domains are mammalian globin-like domains.

45. A non-naturally occuring pseudooligomeric polypeptide comprising four or more globin-like domains.

46. The polypeptide of claim 45, said polypeptide comprising, as an interdomain spacer, a polyproline helix.

47. The polypeptide of claim 45, said polypeptide comprising as an interdomain spacer, a polyaspartate or polyglutamate helix.

48. The polypeptide of claim 45, said polypeptide comprising, as an interdomain spacer, an Artemia linker.

49. The polypeptide of claim 45, said polypeptide comprising, as an interdomain spacers, a helical coiled coil.

50. The polypeptide of claim 45 wherein the globin-like domains are human alpha globin-like domains and the interdomain spacer is about 20-50 Å

.

51. The polypeptide of claim 50 wherein the interdomain spacer is -(Gly)₇ (SEQ ID NO:

32), -(Gly)_1-3 (Ala)₁₂ -(Gly_1-3 (SEQ ID NO;

33) -(Gly)_1-3 (Pro_12-16 -(Gly)_1-3 (SEQ ID NO;

34)- or -(Gly)_1-3 (ASP)_1-30 -(Gly)_1-3 (SEQ ID NO;

35).

52. A non-naturally occuring multimeric hemoglobin-like protein which comprises the polypeptide of claim 45 and one or more additional globin-like domain-bearing polypeptide chains.

53. The protein of claim 52, wherein the crosslinked cysteine is at a mutation site marked on FIG. 2.

54. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 52, wherein each of the globin-like domains is a mammalian globin-like domain.

55. A recombinant DNA molecule comprising a DNA sequence encoding the polypeptide of claim 45.

56. A cell transformed with the molecule of claim 55, said molecule further comprising a promoter, functional in said cell, which is operably linked to said DNA sequence whereby said cell may be caused to express said polypeptide.

57. A method of producing a multimeric hemoglobin-like protein which comprises cultivating the cell of claim 56, said cell further comprising expressible DNA sequences encoding the globin subunits of said protein other than the globin-like domains of said polypeptide, and expressing said polypeptide and said subunits.

58. The polypeptide of claim 45 in which the polypeptide has exactly four globin-like domains.

59. The polypeptide of claim 45, in which the globin-like domains are vertebrate globin-like domains.

60. The polypeptide of claim 45 in which the globin-like domains are mammalian globin-like domains.

61. The polypeptide of claim 45 in which the globin-like domains are human globin-like domains.

62. A non-naturally occuring octameric hemoglobin-like protein which comprises two tetramers, each tetramer comprising two human alpha globin-like domains and two human beta globin-like domains, a cysteine in one tetramer being disulfide bonded to a corresponding cysteine in the second tetramer, the crosslinking of a third tetramer being essentially sterically prevented, with the proviso that the crosslinked cysteine is not at beta 83 (Hemoglobin Ta Li).

63. A method of supplementing the oxygen carrying capacity of blood which comprises administering to a patient a composition comprising the protein of claim 62, wherein each of the globin-like domains is a mammalian globin-like domain.

64. A non-naturally occuring pseudodimeric globin-like polypeptide having two substantially homologous globin-like domains, one of which is mutated to provide an asymmetric crosslinkable cysteine residue, the corresponding residue in the other globin-like domain of said pseudodimeric polypeptide being an amino acid other than cysteine.

65. The polypeptide of claim 64 in which the cysteine residue lies in an alpha globin-like domain.

66. The polypeptide of claim 64 in which the cysteine residue lies in a beta globin-like domain.

67. The polypeptide of claim 64 wherein each of the globin-like domains is a vertebrate globin-like domain.

68. The polypeptide of claim 64 wherein each of the globin-like domains is a mammalian globin-like domain.

69. The polypeptide of claim 64 in which each of the globin-like domains is a human globin-like domain.

70. A recombinant DNA molecule which comprises a DNA sequence encoding the pseudodimeric globin-like polypeptide of claim 64.

71. A cell transformed with the molecule of claim 70, said molecule further comprising a promoter, functional in said cell, which is operably linked to said DNA sequence, whereby said cell may be caused to express said polypeptide.

72. A method of producing a pseudodimeric globin-like polypeptide which comprises cultivating the cell of claim 71 under conditions conducive to expression of said polypeptide.

73. A method of producing a pseudotetrameric hemoglobin-like protein which comprises cultivating the cell of claim 71, said cell further comprising expressible DNA sequences encoding the globin subunits of said protein other than the globin-like domains of said polypeptide, and expressing said polypeptide and said subunits.

74. A method of producing a multimeric hemoglobin-like protein which comprises producing pseudotetrameric hemoglobin-like proteins by the method of claim 73 and then crosslinking the pseudotetrameric proteins.