OLIGOSACCHARIDE MODIFICATION AND LABELING OF PROTEINS

• US 20070190597A1
• Filed: 02/12/2007
• Published: 08/16/2007
• Est. Priority Date: 02/10/2006
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: AGNEW, Brian, Gee, Kyle, Corry, Schuyler
• Granted Patent: US 8,716,033 B2
• Time in Patent Office: Days
• US Class Current: 435/68.1
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 14/47 from mammals
• 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 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 302/01096 Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/534 with radioactive label
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label
• CPC Class Codes: Y10T 436/17 Nitrogen containing

Abstract

The present invention generally relates to methods of functionalizing proteins, particularly antibodies, at oligosaccharide linkages, methods of humanizing antibodies by modifying glycosylation, as well as to novel antibodies linked to modified oligosaccharides. The invention further relates to kits that may be used to produce the antibodies of the invention.

Claims

1. A method of producing a glycomodified protein, comprising

attaching a modified sugar comprising a chemical handle to a GlcNAc residue on a first protein;

and

mixing said first protein with a reporter molecule, carrier molecule or solid support capable of reacting with said chemical handle;

wherein said reporter molecule, carrier molecule or solid support attaches to the protein at said chemical handle, thereby forming a glycomodified protein.

2. The method of claim 1, wherein said first protein is an antibody.

3. The method of claim 1, wherein prior to the attaching step, the method comprises cleaving an oligosaccharide present on a first protein at a GlcNAc-GlcNAc linkage to obtain a protein comprising a GlcNAc residue.

4. The method of claim 3, wherein said oligosaccharide is cleaved using endoglycosidase H cleavage at the GlcNAc-GlcNAc linkage.

5. The method of claim 1, wherein said modified sugar is attached to said GlcNAc using a mutant galactosyl transferase.

6. The method of claim 5, wherein said mutant is a Y289L mutant.

7. The method of claim 1, wherein said modified sugar is an azide-modified sugar and said reporter molecule, carrier molecule or solid support is labeled with alkyne or activated alkyne.

8. The method of claim 7, wherein said azide-modified sugar is UDP-GalNAz.

9. The method of claim 1, further comprising:

cleaving an oligosaccharide present on a second protein at a GlcNAc-GlcNAc linkage to obtain an oligosaccharide having a GlcNAc residue;

binding said oligosaccharide to the reporter molecule, solid support or carrier molecule capable of reacting with said chemical handle.

10. The method of claim 9, wherein the oligosaccharide having a GlcNAc residue is treated with ammonium bicarbonate and said oligosaccharide is attached to an alkyne by a succinimidyl ester.

11. The method of claim 9, wherein said second protein is synthesized in a different cell line or cell type than said first protein.

12. The method of claim 9, wherein said second protein is synthesized in a human cell.

13. The method of claim 1, wherein said modified oligosaccharide comprises a reporter molecule selected from the group consisting of a fluorescent dye, an enzyme, a radiolabel, a metal chelator, or a detectable substrate.

14. The method of claim 1, wherein said modified oligosaccharide comprises a carrier molecule selected from the group consisting of therapeutic agents, DNA, protein, peptides, and sugars.

15. The method of claim 1, wherein the glycomodified protein becomes more or less antigenic as compared to the first protein before it is modified.

16. The method of claim 1, wherein the first protein is derived from a non-human source.

17. The method of claim 16, wherein the glycomodified protein is humanized.

18. The method of claim 3, wherein said oligosaccharide is cleaved using endoglycosidase M cleavage at the GlcNAc-GlcNAc linkage.

19. The method of claim 18, wherein said oligosaccharide is cleaved using endoglycosidase M cleavage at the GlcNAc-GlcNAc linkage, followed by transferring said oligosaccharide to the first protein.

20. The method of claim 1, wherein the attaching step is in a solution substantially free of proteases.

21. A method of labeling an protein by labeling an oligosaccharide attached to said protein, comprising incubating an protein-producing cell in the presence of an unnatural sugar, wherein said unnatural sugar comprises a chemical handle.

22. An antibody comprising a labeled oligosaccharide.