Oligosaccharide modification and labeling of proteins

• US 8,716,033 B2
• Filed: 02/12/2007
• Issued: 05/06/2014
• Est. Priority Date: 02/10/2006
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Agnew, Brian, Corry, Schuyler B., Gee, Kyle R.
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US11/674,136
• Publication Number:
  US 20070190597A1
• Time in Patent Office: 2,640 Days
• Field of Search: None
• US Class Current: 436/544
• 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 an azido-modified galactose to a GlcNAc residue on a first protein having a GlcNAc-GlcNAc linkage;

and

mixing said first protein, having attached thereto said azido-modified galactose with a reporter molecule, carrier molecule or solid support capable of reacting with the azide group of said azido-modified galactose;

wherein said azido-modified galactose is N-azidoacetylgalactosamine (GalNAz) and said reporter molecule, carrier molecule or solid support is labeled with an alkyne or an activated alkyne group;

wherein said reporter molecule, carrier molecule or solid support attaches to the first protein at the azide group of said attached azido-modified galactose through the alkyne or the activated alkyne group, thereby forming the glycomodified protein; and

wherein said attaching of the azido-modified galactose (GalNAz) is carried out by an enzymatic attachment step utilizing a mutant β

-1,4-galactosyl transferase enzyme (Y289L GalT1) and UDP-GalNAz as a substrate, which transfers said azido-modified galactose (GaINAz) from said UDP-GalNAz to said GlcNAc residue of said first 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 first protein is derived from a non-human source.

6. The method of claim 5, wherein said glycomodified protein is humanized.

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

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

9. The method of claim 1, wherein said reporter molecule is selected from the group consisting of a fluorescent dye, an enzyme, a radiolabel, a metal chelator, and a detectable substrate.

10. The method of claim 1, wherein said carrier is selected from the group consisting of therapeutic agents, DNA, protein, peptides, and sugars.