# US 8,785,212 B2 · Oligosaccharide modification and labeling of proteins

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- Number: US 8,785,212 B2
- Document ID: US8785212B2
- Status: Granted
- Kind: B2
- Application number: 13/007,506
- Filed: 2011-01-14
- Published: 2011-08-25
- Granted: 2014-07-22
- Earliest claimed: 2006-02-10
- Assignees: Life Technologies Corporation
- Applicants: Brian Agnew; Kyle Gee; Schuyler Corry
- Inventors: Brian Agnew; Kyle Gee; Schuyler Corry
- Web page: https://patentgrep.com/patent/US8785212B2

## CPC

- C12P 21/005: {Glycopeptides, glycoproteins}
- A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- C07K 1/13: Labelling of peptides
- C07K 14/47: from mammals
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12Y 302/01096: Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- G01N 33/531: Production of immunochemical test materials
- G01N 33/532: Production of labelled immunochemicals
- G01N 33/533: with fluorescent label
- G01N 33/581: {with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)}
- G01N 33/582: {with fluorescent label}
- G01N 33/583: {with non-fluorescent dye label}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/534: with radioactive label
- Y10T 436/17: Nitrogen containing
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## 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

### Claim 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.

### Claim 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.

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 3)

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

### Claim 5 (depends on 3)

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

### Claim 6 (depends on 2)

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

### Claim 7 (depends on 2)

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.

### Claim 8 (depends on 2)

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

### Claim 9 (depends on 2)

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

### Claim 10 (depends on 9)

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.

### Claim 11 (depends on 9)

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.

### Claim 12 (depends on 9)

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.

### Claim 13 (depends on 3)

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

### Claim 14 (depends on 13)

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.

### Claim 15 (depends on 3)

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