IP Library › Granted Patent US 8,097,701
Granted Patent B2
US 8,097,701 · App. 13/163,565 · Granted Jan 17, 2012

Aldehyde tags, uses thereof in site-specific protein modification

Assignee: The Regents of the University of California
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Quick Facts
Patent No.
US 8,097,701
App. No.
13/163,565
Granted
Jan 17, 2012
Kind
B2
Abstract

The invention features compositions and methods for site-specific modification of proteins by incorporation of an aldehyde tag. Enzymatic modification at a sulfatase motif of the aldehyde tag through action of a formylglycine generating enzyme (FGE) generates a formylglycine (FGly) residue. The aldehyde moiety of FGly residue can be exploited as a chemical handle for site-specific attachment of a moiety of interest to a polypeptide.

Claims (91)

1. A non-naturally occurring, recombinant polypeptide comprising a heterologous sulfatase motif having a 2-formylglycine residue, wherein the heterologous sulfatase motif is less than 13 amino acid residues and contains a contiguous sequence of the formula

X 1 (FGly)X 2 Z 2 X 3 R (SEQ ID NO: 1)  (I)

wherein

FGly is a 2-formylglycine residue;

Z 2 is a proline or alanine residue; and

X 1 , X 2 and X 3 are each independently any amino acid.

2. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif

is positioned at a C-terminus of the recombinant polypeptide;

is present in a terminal loop of the recombinant polypeptide;

is, when the recombinant polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop;

is present at an internal site or at an N-terminus of the recombinant polypeptide, and is solvent-accessible when the recombinant polypeptide is folded; and/or

is present at a site of post-translational modification.

3. The non-naturally occurring, recombinant polypeptide of claim 2 , wherein the heterologous sulfatase motif is positioned at an internal sequence of the recombinant polypeptide.

4. The non-naturally occurring, recombinant polypeptide of claim 2 , wherein the heterologous sulfatase motif is positioned at a terminal loop, a C-terminus, or an N-terminus of the recombinant polypeptide.

5. The non-naturally occurring, recombinant polypeptide of claim 2 , wherein the heterologous sulfatase motif is positioned on a solvent-accessible region of the recombinant polypeptide when folded.

6. The non-naturally occurring, recombinant polypeptide of claim 2 , wherein the heterologous sulfatase motif is positioned at a site of post-translational modification of the recombinant polypeptide that is native or non-native to the amino acid sequence of the recombinant polypeptide.

7. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein X 1 , X 2 , and X 3 are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

8. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein X 2 and X 3 are each independently S, T, A, V, G, or C.

9. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein X 1 is L, M, V, S or T and X 2 and X 3 are each independently S, T, A, V, G or C.

10. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein X 1 is L, M, V, S or T.

11. The non-naturally occurring, recombinant polypeptide of claim 10 , wherein the heterologous sulfatase motif is L(FGly)TPSR (SEQ ID NO: 62).

12. The recombinant polypeptide of claim 11 , wherein the non-naturally occurring, recombinant polypeptide comprises an an Fc fragment.

13. The non-naturally occurring, recombinant polypeptide of claim 12 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.

14. The non-naturally occurring, recombinant polypeptide of claim 12 , wherein the antibody is an IgG antibody.

15. The non-naturally occurring, recombinant polypeptide of claim 12 , wherein the antibody is a humanized antibody.

16. The non-naturally occurring, recombinant polypeptide of claim 11 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.

17. The non-naturally occurring, recombinant polypeptide of claim 16 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.

18. The non-naturally occurring, recombinant polypeptide of claim 11 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.

19. The non-naturally occurring, recombinant polypeptide of claim 10 , wherein the heterologous sulfatase motif is selected from M(FGly)TPSR (SEQ ID NO: 63), V(FGly)TPSR (SEQ ID NO: 64), L(FGly)SPSR (SEQ ID NO: 65), L(FGly)APSR (SEQ ID NO: 66), L(FGly)VPSR (SEQ ID NO: 67), and L(FGly)GPSR (SEQ ID NO: 68).

20. The non-naturally occurring, recombinant polypeptide of claim 19 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

21. The non-naturally occurring, recombinant polypeptide of claim 20 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.

22. The non-naturally occurring, recombinant polypeptide of claim 21 , wherein the antibody is an IgG antibody.

23. The non-naturally occurring, recombinant polypeptide of claim 21 , wherein the antibody is a humanized antibody.

24. The non-naturally occurring, recombinant polypeptide of claim 19 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.

25. The non-naturally occurring, recombinant polypeptide of claim 24 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.

26. The non-naturally occurring, recombinant polypeptide of claim 19 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.

27. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

28. The non-naturally occurring, recombinant polypeptide of claim 27 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.

29. The non-naturally occurring, recombinant polypeptide of claim 28 , wherein the antibody is an IgG antibody.

30. The non-naturally occurring, recombinant polypeptide of claim 28 , wherein the antibody is a humanized antibody.

31. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.

32. The non-naturally occurring, recombinant polypeptide of claim 31 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.

33. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.

34. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a blood factor.

35. The non-naturally occurring, recombinant polypeptide of claim 34 , wherein the blood factor is Factor VIII.

36. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a fibroblast growth factor.

37. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a protein vaccine.

38. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is an enzyme.

39. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is less than 12 amino acid residues.

40. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is less than 11 amino acid residues.

41. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is less than 10 amino acid residues.

42. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is less than 9 amino acid residues.

43. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is less than 8 amino acid residues.

44. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is less than 7 amino acid residues.

45. The non-naturally occurring, recombinant polypeptide of claim 25 , wherein the 2-formylglycine residue has been reacted with an aldehyde-reactive group of a moiety of interest to form a modified sulfatase motif in which the moiety of interest is covalently bound.

46. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein X 1 , X 2 , and X 3 are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

47. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein X 1 is L, M, V, S or T.

48. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein X 2 and X 3 are each independently S, T, A, V, G, or C.

49. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein X 1 is L, M, V, S or T and X 2 and X 3 are each independently S, T, A, V, G or C.

50. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein the moiety is a water-soluble polymer, a detectable label, a drug, or an immobilization tag.

51. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein the modified sulfatase motif is positioned at a site of post-translational modification that is native or non-native to the parent of the modified polypeptide.

52. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein the moiety of interest is a drug.

53. The non-naturally occurring, recombinant polypeptide of claim 52 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

54. The non-naturally occurring, recombinant polypeptide of claim 52 , wherein non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.

55. The non-naturally occurring, recombinant polypeptide of claim 52 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.

56. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein the moiety of interest is a toxin.

57. The non-naturally occurring, recombinant polypeptide of claim 56 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

58. The non-naturally occurring, recombinant polypeptide of claim 56 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.

59. The non-naturally occurring, recombinant polypeptide of claim 56 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.

60. The non-naturally occurring, recombinant polypeptide of claim 56 , wherein the moiety of interest is a peptide.

61. The non-naturally occurring, recombinant polypeptide of claim 60 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

62. The non-naturally occurring, recombinant polypeptide of claim 60 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.

63. The non-naturally occurring, recombinant polypeptide of claim 60 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.

64. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

65. The non-naturally occurring, recombinant polypeptide of claim 64 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.

66. The non-naturally occurring, recombinant polypeptide of claim 65 , wherein the antibody is an IgG antibody.

67. The non-naturally occurring, recombinant polypeptide of claim 65 , wherein the antibody is a humanized antibody.

68. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.

69. The non-naturally occurring, recombinant polypeptide of claim 68 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.

70. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.

71. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein X 1 is L, X 2 is T, Z 2 is P, and X 3 is S.

72. The non-naturally occurring, recombinant polypeptide of claim 71 , wherein the moiety of interest is a drug.

73. The non-naturally occurring, recombinant polypeptide of claim 72 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

74. The non-naturally occurring, recombinant polypeptide of claim 73 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.

75. The non-naturally occurring, recombinant polypeptide of claim 74 , wherein the antibody is an IgG antibody.

76. The non-naturally occurring, recombinant polypeptide of claim 74 , wherein the antibody is a humanized antibody.

77. The non-naturally occurring, recombinant polypeptide of claim 72 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.

78. The non-naturally occurring, recombinant polypeptide of claim 77 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.

79. The non-naturally occurring, recombinant polypeptide of claim 72 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.

80. The non-naturally occurring, recombinant polypeptide of claim 45 , wherein X 1 is L, M, or V, Z 2 is P, and X 2 and X 3 are each independently S, T, A, V, G or C.

81. The non-naturally occurring, recombinant polypeptide of claim 80 , wherein the moiety of interest is a drug.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 29, 2011
From: CARRICO, ISAAC S.; CARLSON, BRIAN L.; WU, PENG; BERTOZZI, CAROLYN
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 026821/0036 →
Continuity (3)
Division 11857385 · Sep 18, 2007
Provisional Application 60846644 · Sep 21, 2006
Related Publication 20110287041A1 · Nov 24, 2011