Aldehyde tags, uses thereof in site-specific protein modification

• US 7,985,783 B2
• Filed: 09/18/2007
• Issued: 07/26/2011
• Est. Priority Date: 09/21/2006
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Wu, Peng, Carlson, Brian L., Carrico, Isaac S., Bertozzi, Carolyn
• Primary Examiner(s): Kim; Alexander D
• Application Number:
  US11/857,385
• Publication Number:
  US 20080187956A1
• Time in Patent Office: 1,407 Days
• Field of Search: None
• US Class Current: 522/87
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 38/215 IFN-beta
• CPC Class Codes: A61K 38/37 Factors VIII
• CPC Class Codes: A61K 38/45 Transferases (2)
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 1/006 of peptides containing derivatised side chain amino acids
• CPC Class Codes: C07K 1/1072 by covalent attachment of residues or functional groups
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/35 from Mycobacteriaceae (F)
• CPC Class Codes: C07K 14/565 IFN-beta
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 9/13 transferring sulfur containing groups (2.8)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/005 : Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 208/02 : Sulfotransferases (2.8.2)

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

1. A method for modifying a polypeptide, the method comprising:

contacting a non-naturally occurring, recombinant polypeptide comprising a heterologous, converted sulfatase motif generated by reaction with a formylglycine-generating enzyme (FGE) with a reactive partner comprising an aldehyde-reactive group and a moiety of interest, wherein the heterologous, converted sulfatase motif is less than 13 amino acid residues and contains a contiguous sequence of the formula;

2. The method of claim 1, wherein the heterologous, converted sulfatase motif is positioned in the non-naturally occurring, recombinant polypeptide:

at an internal sequence of the polypeptide;

at a terminal loop, a C-terminus, or an N-terminus of the recombinant polypeptide;

at a solvent-accessible region of the recombinant polypeptide when folded; and

/or

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.

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

4. The method 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 method of claim 2, wherein the heterologous sulfatase motif is positioned on a solvent-accessible region of the recombinant polypeptide when folded.

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

7. The method of claim 1, wherein X₁, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid or a polar, uncharged amino acid.

8. The method of claim 1, wherein X₁ is L, M, V, S or T.

9. The method of claim 1, wherein X₂ and X₃ are each independently S, T, A, V, G or C.

10. The method of claim 9, wherein X₁ is L, M, V, S or T.

11. The method of claim 10, wherein the converted sulfatase motif is L(FGly)TPSR (SEQ ID NO:

62).

12. The method of claim 10, wherein the converted 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).

13. The method of claim 1, wherein X₁ is L, M, V, S or T and X₂ and X₃ are each independently S, T, A, V, G or C.

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

15. The method of claim 14, wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.

16. The method of claim 15, wherein the non-naturally occurring, recombinant polypeptide is an antibody.

17. The method of claim 16, wherein the antibody is an IgG antibody.

18. The method of claim 16, wherein the antibody is a humanized antibody.

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

20. The method of claim 19, wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.

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

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

23. The method of claim 22, wherein the blood factor is Factor VIII.

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

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

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

27. The method of claim 1, wherein the moiety of interest is a drug.

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

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

30. The method of claim 27, wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.

31. The method of claim 1, wherein the moiety of interest is a toxin.

32. The method of claim 31, wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

33. The method of claim 31, wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.

34. The method of claim 31, wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.

35. The method of claim 1, wherein the moiety of interest is a peptide.

36. The method of claim 35, wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

37. The method of claim 35, wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.

38. The method of claim 35, wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.

39. The method of claim 1, wherein the aldehyde-reactive group of the reactive partner comprises an aminooxy.

40. The method of claim 1, wherein the aldehyde-reactive group of the reactive partner comprises a hydrazide.

41. The method of claim 1, wherein the aldehyde-reactive group of the reactive partner comprises a thiosemicarbazide.

42. The method of claim 1, wherein the heterologous, converted sulfatase motif is less than 12 amino acid residues.

43. The method of claim 1, wherein the heterologous, converted sulfatase motif is less than 11 amino acid residues.

44. The method of claim 1, wherein the heterologous, converted sulfatase motif is less than 10 amino acid residues.

45. The method of claim 1, wherein the heterologous, converted sulfatase motif is less than 9 amino acid residues.

46. The method of claim 1, wherein the heterologous, converted sulfatase motif is less than 8 amino acid residues.

47. The method of claim 1, wherein the heterologous, converted sulfatase motif is less than 7 amino acid residues.