Aldehyde tags, uses thereof in site-specific protein modification

Patent metadata

Field	Value
Publication number	US-10150806-B2
Application number	US-201615238158-A
Country	US
Kind code	B2
Filing date	Aug 16, 2016
Priority date	Sep 21, 2006
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

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.

First claim

That which is claimed is:

1. A method comprising:
administering a polypeptide to a subject, wherein the polypeptide is a modified non-naturally occurring, recombinant polypeptide comprising a heterologous sulfatase motif having a 2-formylglycine residue covalently attached to a moiety of interest, 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: 97)

wherein
FGly′ is the 2-formylglycine residue covalently attached to the moiety of interest;
Z 2 is a proline or alanine residue;
X 1 is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the polypeptide, X 1 is present; and
X 2 and X 3 are each independently any amino acid.

2. The method of claim 1 , wherein
the heterologous sulfatase motif is positioned at a C-terminus of the modified non-naturally occurring, recombinant polypeptide;
the modified non-naturally occurring, recombinant polypeptide comprises a terminal loop and the heterologous sulfatase motif is present in the terminal loop of the modified non-naturally occurring, recombinant polypeptide;
the heterologous sulfatase motif is, when the modified non-naturally occurring, recombinant polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop;
the heterologous sulfatase motif is present at an internal site or at the N-terminus of the modified non-naturally occurring, recombinant polypeptide, and is solvent-accessible when the polypeptide is folded; or
the heterologous sulfatase motif is present at a site of post-translational modification.

3. The method of claim 1 , wherein the heterologous sulfatase motif is positioned in the modified non-naturally occurring, recombinant polypeptide at a site of post-translational modification of that is native or non-native to the parent of the modified non-naturally occurring, recombinant polypeptide.

4. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide is selected from the group consisting of an Fc fragment, an antibody, an antigen-binding fragment of an antibody, a blood factor, a fibroblast growth factor, a protein vaccine, and an enzyme.

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

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

7. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide is an antibody.

8. The method of claim 7 , wherein the antibody is an IgG antibody.

9. The method of claim 7 , wherein the antibody is a humanized antibody.

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

11. The method of claim 10 , wherein the modified non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.

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

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

14. The method of claim 13 , wherein the blood factor is Factor VIII.

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

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

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

18. The method of claim 1 , wherein the moiety of interest is a water-soluble polymer, a detectable label, a drug, a toxin, a peptide, or an immobilization tag.

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

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

21. The method of claim 1 , wherein X 1 , when present, X 2 , and X 3 are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

22. The method of claim 1 , wherein the X 1 , when present, is L, M, V, S or T.

23. The method of claim 1 , wherein X 2 and X 3 are each independently S, T, A, V, or C.

24. The method of claim 1 , wherein the heterologous sulfatase motif is selected from M(FGly′)TPSR (SEQ ID NO: 123), V(FGly′)TPSR (SEQ ID NO: 124), L(FGly′)SPSR (SEQ ID NO: 125), L(FGly′)APSR (SEQ ID NO: 126), L(FGly′)VPSR (SEQ ID NO: 127), and L(FGly′)GPSR (SEQ ID NO: 128).

25. The method of claim 1 , wherein the heterologous sulfatase motif is L(FGly′)TPSR (SEQ ID NO: 129).

26. The method of claim 1 , wherein the heterologous sulfatase motif is less than 12 amino acid residues and greater than or equal to 5 amino acid residues.

27. The method of claim 1 , wherein the heterologous sulfatase motif is less than 11 amino acid residues and greater than or equal to 5 amino acid residues.

28. The method of claim 1 , wherein the heterologous sulfatase motif is less than 10 amino acid residues and greater than or equal to 5 amino acid residues.

29. The method of claim 1 , wherein the heterologous sulfatase motif is less than 9 amino acid residues and greater than or equal to 5 amino acid residues.

30. The method of claim 1 , wherein the heterologous sulfatase motif is less than 8 amino acid residues and greater than or equal to 5 amino acid residues.

31. The method of claim 1 , wherein the heterologous sulfatase motif is less than 7 amino acid residues and greater than or equal to 5 amino acid residues.

Assignees

• Univ California

Inventors

• Carrico Isaac S
• Carlson Brian L
• Wu Peng
• Bertozzi Carolyn Ruth

Classifications

• C07K2317/40

  characterized by post-translational modification · CPC title

• C12P21/005

  Glycopeptides, glycoproteins · CPC title

• A61K38/45

  Transferases (2) · CPC title

• A61P37/04

  Immunostimulants · CPC title

• C07K16/46

  Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00) · CPC title