Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity

• US 6,602,684 B1
• Filed: 04/20/1999
• Issued: 08/05/2003
• Est. Priority Date: 04/20/1998
• Status: Expired due to Term

• Current Assignee: Roche Glycart AG (Roche Holding AG)
• Original Assignee: Glycart Biotechnology AG (Roche Holding AG)
• Inventors: Umaña, Pablo, Jean-Mairet, Joël, Bailey, James E.
• Primary Examiner(s): Yucel, Remy
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US09/294,584
• Time in Patent Office: 1,568 Days
• Field of Search: 435/69.1, 435/320.1, 435/455, 536/23.1, 536/24.1
• US Class Current: 435/69.1
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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 16/2887 against CD20
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/3053 Skin, nerves, brain
• 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: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: Y10S 977/802 Virus-based particle

Abstract

The present invention relates to the field glycosylation engineering of proteins. More particular, the present invention is directed to the glycosylation engineering of proteins to provide proteins with improved therapeutic properties, e.g., antibodies, antibody fragments, or a fusion protein that includes a region equivalent to the Fc region of an immunoglobulin, with enhanced Fc-mediated cellular cytotoxicity.

Claims

1. A method for producing a polypeptide having increased Fc-mediated cellular cytotoxicity in a host cell, comprising:

(a) culturing a host cell engineered to express at least one nucleic acid encoding β

(1,4)-N-acetylglucosaminyltransferase III (GnT III) under conditions which permit the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment, and a fusion protein that includes the Fc region of an immunoglobulin, wherein said GnT III is expressed in an amount sufficient to modify the oligosaccharides in the Fc region of said polypeptide produced by said host cell and wherein said polypeptide has increased Fc-mediated cellular cytotoxicity as a result of said modification; and

(b) isolating said polypeptide having increased Fc-mediated cellular cytotoxicity.

2. The method of claim 1, wherein in step (a), said host cell comprises at least one nucleic acid encoding a whole antibody.

3. The method of claim 1, wherein in step (a), said host cell comprises at least one nucleic acid encoding an antibody fragment.

4. The method of claim 1, wherein in step (a), said host cell comprises at least one nucleic acid encoding a fusion protein comprising a glycosylated Fc region of an immunoglobulin.

5. The method of claim 1, wherein the expression level of glycosyl transferase GnT III produces an antibody molecule, antibody fragment, or a fusion protein that includes the Fc region of an immunoglobulin having increased Fc-mediated cellular cytotoxicity at a higher level than the Fc-mediated cellular cytotoxicity obtained from a different expression level of the same glycosyl transferase GnT III gene.

6. The method of claim 1, wherein said host cell further comprises a nucleic acid encoding a glycosidase.

7. The method of claim 1, wherein the expression level of glycosyl transferase GnTIII is sufficient to form bisected oligosaccharides in the Fc region of said polypeptide.

8. The method of claim 7, wherein the proportion of bisected oligosaccharides in the Fc region to total oligosaccharides in the Fc region is at least 45 percent.

9. The method of claim 7, wherein said bisected oligosaccharides are bisected, complex oligosaccharides.

10. A method for producing a polypeptide having increased Fc-mediated cellular cytotoxicity in a host cell, comprising:

(a) culturing a host cell engineered to express at least one nucleic acid encoding β

(1,4)-N-acetylglucosaminyltransferase III (GnT III) under conditions which permit the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment, and a fusion protein that includes a Fc region of an immunoglobulin and an antigen binding region, wherein said GnTIII is expressed in an amount sufficient to modify oligosaccharides in the Fc region of said polypeptide produced by said host cell, and wherein said polypeptide has increased Fc-mediated cellular cytotoxicity as a result of said modification; and

(b) isolating said polypeptide having increased Fc-mediated cellular cytotoxicity;

wherein said increased Fc-mediated cellular cytotoxicity is determined by an increase in antibody-dependent cellular cytotoxicity as measured in the following standard in vitro assay which uses viable target cells that are known to express a target antigen recognized by the antigen-binding region of said polypeptide, and uses as effector cells human peripheral blood mononuclear cells (PBMCs), said standard in vitro assay comprising the steps of;

(i) labeling said target cells with the fluorescent dye Calcein AM as a marker for cell integrity;

(ii) obtaining a first portion of said labeled target cells and dividing said first portion into multiple equal subportions of said labeled target cells;

(iii) obtaining second and third portions of said labeled target cells having the same number of labeled target cells as said multiple equal subportions;

(iv) mixing each said multiple equal subportion of said labeled target cells with a different concentration of polypeptide in a multi-well assay plate, each concentration being tested in triplicate and said different concentrations of polypeptide chosen to give different percentages of specific lysis;

(v) mixing said second portion of said labeled target cells with a detergent that lyses said labeled target cells in said multi-well assay plate to provide a total lysis control portion;

(vi) mixing said third portion of said labeled target cells with antibody-free culture medium in said multi-well assay plate to provide a spontaneous release control portion;

(vii) incubating said multi-well assay plate containing said multiple equal subportions, total lysis control portion, and spontaneous release control portion for 1 hr;

(viii) adding effector cells to each of said multiple equal subportions, total lysis control portion, and spontaneous release control portion in said multi-well assay plate to yield an effector cell;

target cell ratio of 19;

1 and mixing;

(ix) incubating said multi-well assay plate in an incubator under 5% CO₂ atmosphere at 37°

C. for 16 hrs;

(x) discarding the cell free supernatant from each well of said multi-well assay plate;

(xi) washing said labeled target cells in said multi-well assay plate with buffered saline solution;

(xii) lysing said labeled target cells in nonionic detergent t-octylphenoxypolyethoxyethanol at a final concentration of 0.1% (v/v);

(xiii) measuring the experimentally retained fluorescence (EF) of said target cells with a fluorometer;

wherein the percentage of specific cell lysis for each polypeptide concentration is calculated according to the formula (SR-EF)/(SR-MR)×

100, where EF is average fluorescence measured for a given polypeptide concentration, MR is the average fluorescence measured for said total lysis control portion, and SR is the average fluorescence measured for said spontaneous release control portion, and wherein an increase in antibody-dependent cellular cytotoxicity is measured as either an increase in the maximum percentage of specific lysis observed within the polypeptide concentration range tested, and/or a reduction in the concentration of polypeptide required to achieve one half of the maximum percentage of specific lysis observed within the polypeptide concentration range tested.