Oligosaccharide modification and labeling of proteins

• US 8,785,212 B2
• Filed: 01/14/2011
• Issued: 07/22/2014
• 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, Gee, Kyle, Corry, Schuyler
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US13/007,506
• Publication Number:
  US 20110207171A1
• Time in Patent Office: 1,285 Days
• 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 labeling a protein by labeling an oligosaccharide attached to said protein, comprising:

incubating a protein-producing cell in the presence of an unnatural sugar, wherein the unnatural sugar comprises an azide group, to provide a modified protein, wherein the modified protein produced by the protein-producing cell is attached to an oligosaccharide that comprises the unnatural sugar, wherein the unnatural sugar comprises GalNAz, GlcNAz, Ac₄GalNAz, or Ac₄GlcNAz; and

attaching the modified protein to a label that comprises an alkyne group or an activated alkyne group by contacting the modified protein with the label under a condition such that a covalent bond is formed between the alkyne or the activated alkyne group of the label and the azide group of the unnatural sugar, thereby labeling the protein.

2. A method of metabolically modifying a protein by modifying an oligosaccharide group attached to the protein, comprising:

incubating a protein-producing cell in the presence of an unnatural substance, wherein the unnatural substance comprises an azide group, to provide a modified protein, wherein the modified protein produced by the protein-producing cell is attached to an oligosaccharide that comprises the unnatural substance, wherein the unnatural substance comprises an unnatural sugar selected from GalNAz, GlcNAz, Ac₄GalNAz, and Ac₄GlcNAz; and

attaching the modified protein to a label that comprises an alkyne group or an activated alkyne group by contacting the modified protein with the label under a condition such that a covalent bond is formed between the alkyne or the activated alkyne group of the label and the azide group of the unnatural sugar, thereby modifying the protein.

3. The method of claim 2, wherein the modified protein produced by the cell is a modified recombinant protein or a modified antibody.

4. The method of claim 3, wherein the antibody comprises an IgG.

5. The method of claim 3, wherein the antibody comprises a monoclonal antibody, recombinant antibody, or a fragment thereof which is immunologically active.

6. The method of claim 2, wherein the protein-producing cell comprises a hybridoma cell or a cell producing a recombinant protein.

7. The method of claim 2, wherein the protein-producing cell is a murine cell, a plant cell, a goat cell, a rabbit cell, a yeast cell, or a Chinese hamster ovary cell.

8. The method of claim 2, wherein the step of attaching results in the formation of a covalent bond.

9. The method of claim 2, wherein the label comprises a reporter molecule, carrier molecule or solid support.

10. The method of claim 9, wherein the reporter molecule comprises a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme, or a radioisotope.

11. The method of claim 9, wherein the carrier molecule comprises an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, a synthetic polymer, a polymeric microparticle, a biological cell, cellular components, an ion chelating moiety, an enzymatic substrate, a virus, an antibody or fragment thereof, an antigen, an avidin, a streptavidin, a biotin, a dextran, an IgG binding protein, a fluorescent protein, agarose, or a non-biological microparticle.

12. The method of claim 9, wherein the solid support comprises an aerogel, a hydrogel, a resin, a bead, a biochip, a microfluidic chip, a silicon chip, a multi-well plate, a membrane, a conducting metal, a nonconducting metal, a glass, a magnetic support, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, a poly(acrylate), a polystyrene, a poly(acrylamide), a polyol, an agarose, an agar, a cellulose, a dextran, a starch, a FICOLL, a heparin, a glycogen, an amylopectin, a mannan, an inulin, a nitrocellulose, a diazocellulose, a polyvinylchloride, a polypropylene, a polyethylene, a nylon, a latex bead, a magnetic bead, a paramagnetic bead, a superparamagnetic bead, or a starch.

13. The method of claim 3, wherein the label comprises a therapeutic moiety.

14. The method of claim 13, wherein the therapeutic moiety comprises taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, a glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, or analogs or homologs thereof;

or, an antimetabolite, an alkylating agent, an anthracycline, an antibiotic, an anti-mitotic agent, an antimetabolite, an alkylating agent, an anthracycline, an antibiotic, an anti-mitotic agent, abrin, ricin A, pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, γ

-interferon, α

-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, interleukin-1, interleukin-2, interleukin-6, granulocyte macrophage colony stimulating factor, or granulocyte colony stimulating factor.

15. The method of claim 3, further comprising the step of isolating the modified antibody.