Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity

Abstract

The present invention relates to the field of glycosylation engineering of proteins. More particularly, the present invention relates to glycosylation engineering to generate proteins with improved therapeutic properties, including antibodies with increased antibody-dependent cellular cytotoxicity.

What is claimed is:  
     
         1 . A host cell engineered to produce a polypeptide having increased Fc-mediated cellular cytotoxicity by expression of at least one nucleic acid encoding β(1,4)-N-acetylglucosaminyltransferase III (GnT III), wherein said polypeptide produced by said host cell is selected from the group consisting of a whole antibody molecule, an antibody fragment, and a fusion protein which includes a region equivalent to the Fc region of an immunoglobulin, and wherein said GnT III is expressed in an amount sufficient to increase the proportion of said polypeptides carrying bisected hybrid oligosaccharides or galactosylated complex oligosaccharides or mixtures thereof in the Fc region relative to polypeptides carrying bisected complex oligosaccharides in the Fc region.  
     
     
         2 . The host cell of  claim 1 , wherein said polypeptide is IgG or a fragment thereof.  
     
     
         3 . The host cell of  claim 1 , wherein said polypeptide is IgG1 or a fragment thereof.  
     
     
         4 . The host cell of  claim 1 , wherein said polypeptide is a fusion protein that includes a region equivalent to the Fc region of a human IgG.  
     
     
         5 . The host cell of  claim 1 , wherein a nucleic acid molecule comprising at least one gene encoding GnTIII has been introduced into said host cell.  
     
     
         6 . The host cell of  claim 1 , wherein said host cell has been engineered such that an endogenous GnT III gene is activated.  
     
     
         7 . The host cell of  claim 6 , wherein said endogenous GnTIII has been activated by insertion of a DNA element which increases gene expression into the host chromosome.  
     
     
         8 . The host cell of  claim 6 , wherein said host cell has been selected to carry a mutation triggering expression of an endogenous GnTIII.  
     
     
         9 . The host cell of  claim 8 , wherein said host cell is the CHO cell mutant lec 10.  
     
     
         10 . The host cell of  claim 1 , wherein said host cell is a CHO cell, a BHK cell, a NS0 cell, a SP2/0 cell, a YO myeloma cell, a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell or a hybridoma cell.  
     
     
         11 . The host cell of  claim 10 , wherein said polypeptide is an anti-CD20 antibody.  
     
     
         12 . The host cell of  claim 11 , wherein said anti-CD20 antibody is IDEC-C2B8.  
     
     
         13 . The host cell of  claim 10 , wherein said host cell is a SP2/0 cell.  
     
     
         14 . The host cell of  claim 13 , wherein said antibody is the chimeric anti-human renal cell carcinoma monoclonal antibody chG250.  
     
     
         15 . The host cell of  claim 5 , wherein said at least one gene encoding GnTIII has been introduced into said host cell chromosome.  
     
     
         16 . The host cell of  claim 6 , wherein said endogenous GnTIII has been activated by insertion of a promoter element, a transposon, or a retroviral element into the host cell chromosome.  
     
     
         17 . The host cell of  claim 1 , further comprising at least one transfected nucleic acid encoding an antibody molecule, an antibody fragment, or a fusion protein that includes a region equivalent to the Fc region of an immunoglobulin.  
     
     
         18 . The host cell of  claim 1 , wherein said at least one nucleic acid encoding a GnTIII is operably linked to a constitutive promoter element.  
     
     
         19 . The host cell of  claim 17 , wherein said host cell comprises at least one transfected nucleic acid encoding an anti-CD20 antibody, the chimeric anti-human neuroblastoma monoclonal antibody chCE7, the chimeric anti-human renal cell carcinoma monoclonal antibody chG250, the chimeric anti-human colon, lung, and breast carcinoma monoclonal antibody ING-1, the humanized anti-human 17-1A antigen monoclonal antibody 3622W94, the humanized anti-human colorectal tumor antibody A33, the anti-human melanoma antibody directed against GD3 ganglioside R24, the chimeric anti-human squamous-cell carcinoma monoclonal antibody SF-25, an anti-human EGFR antibody, an anti-human EGFRvIII antibody, an anti-human PSMA antibody, an anti-human PSCA antibody, an anti-human CD22 antibody, an anti-human CD30 antibody, an anti-human CD33 antibody, an anti-human CD38 antibody, an anti-human CD40 antibody, an anti-human CD45 antibody, an anti-human CD52 antibody, an anti-human CD138 antibody, an anti-human BLA-DR variant antibody, an anti-human EpCAM antibody, an anti-human CEA antibody, an anti-human MUC1 antibody, an anti-human MUC1 core protein antibody, an anti-human aberrantly glycosylated MUC1 antibody, an antibody against human fibronectin variants containing the ED-B domain, or an anti-human HER2/neu antibody.  
     
     
         20 . A method for producing a polypeptide in a host cell comprising culturing the host cell of any one of claims  1 - 19  under conditions which permit the production of said polypeptide having increased Fc-mediated cellular cytotoxicity.  
     
     
         21 . The method of  claim 20 , further comprising isolating said polypeptide having increased Fc-mediated cellular cytotoxicity.  
     
     
         22 . The method of  claim 20 , wherein said host cell comprises at least one nucleic acid encoding a fusion protein comprising a region equivalent to a Fc region of an immunoglobulin.  
     
     
         23 . The method of  claim 20 , wherein greater than 50% of the oligosaccharides in the Fc region of said polypeptides are bisected.  
     
     
         24 . The method of  claim 20 , wherein greater than 70% of the oligosaccharides in the Fc region of said polypeptides are bisected.  
     
     
         25 . The method of  claim 20 , wherein the proportion of bisected hybrid oligosaccharides or galactosylated complex oligosaccharides or mixtures thereof in the Fc region is greater than the proportion of bisected complex oligosaccharides in the Fc region of said polypeptides.  
     
     
         26 . The method of  claim 20 , wherein said polypeptide is the anti-CD20 antibody IDEC-C2B8 and the IDEC-C2B8 antibodies produced by said host cell have a glycosylaton profile, as analyzed by MALDI/TOF-MS, that is substantially equivalent to that shown in FIG. 2E.  
     
     
         27 . The method of  claim 20 , wherein said polypeptide is the chG250 monoclonal antibody and the chG250 antibodies produced by said host cell have a glycosylaton profile, as analyzed by MALDI/TOF-MS, that is substantially equivalent to that shown in FIG. 7D.  
     
     
         28 . An antibody having increased antibody dependent cellular cytotoxicity (ADCC) produced by the method of  claim 21 .  
     
     
         29 . The antibody of  claim 28 , wherein said antibody is selected from the group consisting of IDEC-C2B8, chCE7, ch-G250, a humanized anti-HER2 monoclonal antibody, ING-1, 3622W94, SF-25, A33, and R24.  
     
     
         30 . An antibody fragment that includes a region equivalent to the Fc region of an immunoglobulin, having increased Fc-mediated cellular cytotoxicity produced by the method of  claim 21 .  
     
     
         31 . A fusion protein that includes a region equivalent to the Fc region of an immunoglobulin and having increased Fc-mediated cellular cytotoxicity produced by the method of  claim 21 .  
     
     
         32 . A pharmaceutical composition comprising the antibody of  claim 28  and a pharmaceutically acceptable carrier.  
     
     
         33 . A pharmaceutical composition comprising the antibody fragment of  claim 30  and a pharmaceutically acceptable carrier.  
     
     
         34 . A pharmaceutical composition comprising the fusion protein of  claim 31  and a pharmaceutically acceptable carrier.  
     
     
         35 . A method for the treatment of cancer comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims  32 - 34  to a patient in need thereof.  
     
     
         36 . An improved method for disease treatment based on B-cell depletion comprising administering a therapeutically effective amount of antibody to a human subject in need thereof, the improvement comprising administering a therapeutically effective amount of an antibody produced by the method of  claim 28 .  
     
     
         37 . The improved method of  claim 36 , wherein said antibody is an anti-CD20 monoclonal antibody.  
     
     
         38 . The improved method of  claim 37 , wherein said anti-CD20 antibody is IDEC-C2B8.