Aldehyde tags, uses thereof in site-specific protein modification

Patent metadata

Field	Value
Publication number	US-10745464-B2
Application number	US-201816169292-A
Country	US
Kind code	B2
Filing date	Oct 24, 2018
Priority date	Sep 21, 2006
Publication date	Aug 18, 2020
Grant date	Aug 18, 2020

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 nucleic acid molecule comprising a nucleotide sequence encoding a non-naturally occurring, recombinant polypeptide comprising a heterologous sulfatase motif capable of being converted by a formylglycine generating enzyme to have a 2-formylglycine residue, wherein the heterologous sulfatase motif is less than 13 amino acid residues and contains a contiguous sequence of the formula:

(I)

(SEQ ID NO: 1)

X 1 (FGly)X 2 Z 2 X 3 R

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 nucleic acid molecule of claim 1 , wherein
the heterologous sulfatase motif is positioned at a C-terminus of the recombinant polypeptide;
the non-naturally occurring, recombinant polypeptide comprises a terminal loop and the heterologous sulfatase motif is present in the terminal loop of the recombinant polypeptide;
the heterologous sulfatase motif is, when the 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 an N-terminus of the recombinant polypeptide, and is solvent-accessible when the recombinant polypeptide is folded; or
the heterologous sulfatase motif is present at a site of post-translational modification.

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

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

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

7. The nucleic acid molecule 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 nucleic acid molecule of claim 1 , wherein X 2 and X 3 are each independently S, T, A, V, G, or C.

9. The nucleic acid molecule 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 nucleic acid molecule of claim 1 , wherein X 1 is L, M, V, S or T.

11. The nucleic acid molecule of claim 10 , wherein the heterologous sulfatase motif is L(FGly)TPSR (SEQ ID NO: 62).

12. The nucleic acid molecule 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).

13. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.

14. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.

15. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.

16. The nucleic acid molecule of claim 15 , wherein the antibody is an IgG antibody.

17. The nucleic acid molecule of claim 15 , wherein the antibody is a humanized antibody.

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

19. The nucleic acid molecule of claim 18 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.

20. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.

21. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a blood factor.

22. The nucleic acid molecule of claim 21 , wherein the blood factor is Factor VIII.

23. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a fibroblast growth factor.

24. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a protein vaccine.

25. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is an enzyme.

26. The nucleic acid molecule 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 nucleic acid molecule 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 nucleic acid molecule 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 nucleic acid molecule 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 nucleic acid molecule 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 nucleic acid molecule 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

• C07K1/006 Primary

  of peptides containing derivatised side chain amino acids · CPC title

• A61K39/00

  Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) · CPC title

• A61K38/215

  IFN-beta · CPC title

• C12N9/13

  transferring sulfur containing groups (2.8) · CPC title

• C07K16/46

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