Modified Host Cells and Uses Thereof

Abstract

The present disclosure provides compositions and methods comprising cells producing glycoproteins with variant glycosylation patterns. The methods and compositions may be used in producing antibodies and proteins of therapeutic value.

1 - 17 . (canceled) 
     
     
         18 . An N-linked glycan comprising one glucose molecule, four mannose molecules, and two N-acetylglycosamine molecules. 
     
     
         19 . The N-linked glycan of  claim 18 , wherein glucose is located at a terminal end of N-linked glycan, and wherein N-linked glycan optionally comprises one or more fucose molecules. 
     
     
         20 . The N-linked glycan of  claim 18  having a structure of formula (I) 
       
         
           
           
               
               
           
         
       
     
     
         21 . The N-linked glycan of  claim 18  having a structure of formula (II) 
       
         
           
           
               
               
           
         
       
     
     
         22 . An isolated glycoprotein that contains an N-glycan of any one of  claims 18 - 21 . 
     
     
         23 . The glycoprotein of  claim 22  wherein glycoprotein is an enzyme. 
     
     
         24 . The glycoprotein of  claim 22  wherein glycoprotein is an antibody. 
     
     
         25 . The glycoprotein of  claim 24  wherein N-linked glycan is attached to an Fc region of antibody. 
     
     
         26 . The glycoprotein of  claim 24  wherein antibody binds to a cancer antigen. 
     
     
         27 . The glycoprotein of  claim 26  wherein cancer antigen is selected from group consisting of HER2, Immunoglobulin epsilon Fc receptor II, Alk-1, CD20, EGF receptor, VEGF receptor, FGF receptor, NGF receptor, PDGF receptor, EpCam, CD3, CD4, CD11a, CD19, CD22, CD30, CD33, CD38, CD40, CD51, CD55, CD80, CD95, CCR2, CCR3, CCR4, CCR5, CTLA-4, Mucin 1, Mucin 16, Endoglin, Mesolin receptor, Nogo receptor, folate receptor, CXCR4, insulin-like growth factor receptor, Ganglioside GD3, and alpha and beta Integrins. 
     
     
         28 . The glycoprotein of  claim 24  wherein antibody is produced by a modified host cell which produces a substantially homogeneous population of N-linked glycans. 
     
     
         29 . The glycoprotein of  claim 24  wherein antibody has an increased ADCC (antibody-dependent cellular cytotoxicity) activity as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012). 
     
     
         30 . The glycoprotein of  claim 24  wherein antibody has an increased binding affinity to FcγRIIIA receptor as compared to a corresponding antibody produced by an unmodified CHO cell clone, CHO-K1 (ATCC #CCL-61 and CRL-9618) or CHO-DG44 (Invitrogen #12609-012). 
     
     
         31 . The glycoprotein of  claim 24  wherein antibody is an inhibitory antibody. 
     
     
         32 . The glycoprotein of  claim 24  wherein antibody is an IgG antibody. 
     
     
         33 . A method of producing a modified glycoprotein comprising:
 (a) providing a heterologous polynucleotide sequence that encodes said modified glycoprotein; and   (b) causing said modified glycoprotein to be expressed in a host cell, wherein host cell produces N-linked glycan having a variant glycosylation pattern characterized by a change in level of one or more sugar moieties selected from group consisting of glucose, galactose, mannose, and glucosamine.   
     
     
         34 . The method of  claim 33 , wherein said host cell is maintained in a serum free medium. 
     
     
         35 . The method of  claim 33 , wherein said host cell is maintained in a suspension culture. 
     
     
         36 . The method of  claim 33 , wherein said modified glycoprotein is an antibody. 
     
     
         37 . The method of  claim 33 , wherein host cell produces N-linked glycan having a structure of formula (I) or (II):