Gylocosylated humanized B-cell specific antibodies

Abstract

A humanized specific monoclonal antibody or antibody fragment, especially a B-cell specific antibody or antibody fragment, is engineered to contain a glycosylation site in the non-Fc constant region. The glycosylated antibody is useful for diagnosis and/or therapy whenever a targeting antibody or fragment is used, especially for B-cell malignancies. The carbohydrate moiety allows conjugation of labeling or therapeutic agents of increased size, without affecting the binding affinity or specificity of the antibody.

We claim:  
     
         1 . A method of producing an antibody or antigen-binding fragment thereof comprising: 
 expressing said antibody or antigen-binding fragment thereof which is engineered to contain a glycosylation site in the non-Fc constant heavy chain region, wherein said antibody or antigen-binding fragment is glycosylated in the CH1 region, or in the constant light chain region, wherein genes encoding said heavy chain and light chain regions have been engineered with a mutation such that a glycosylation site is created in the CH1 region gene or the constant light chain gene, and operably linked to expression control elements in an expression vector, in a cell that allows glycosylation; and    producing said antibody or antibody fragment glycosylated in the CH1 region or the light chain constant region in said cell.    
     
     
         2 . The method of  claim 1 , wherein said expression vector comprises an amplifiable dihydrofolate reductase (dhfr) gene.  
     
     
         3 . The method of  claim 2 , wherein said expression vector is pdHL2.  
     
     
         4 . The method of  claim 3 , wherein said cell is a SP2/0 myeloma cell.  
     
     
         5 . The method of  claim 1 , wherein the antibody or fragment thereof comprises a humanized antibody or antigen-binding fragment thereof.  
     
     
         6 . The method of  claim 1 , wherein the antibody or fragment thereof comprises a humanized B-cell specific antibody or antigen-binding fragment thereof.  
     
     
         7 . The method of  claim 6 , wherein said glycosylation is located on a site selected from the group consisting of the HCN1, HCN2, HCN3, HCN4, and HCN5 sites (SEQ ID NOS: 10-14) of FIG. 12.  
     
     
         8 . The method of  claim 7 , wherein said glycosylation site is the HCN5 site (SEQ ID NO: 10) of FIG. 12.  
     
     
         9 . The method of  claim 7 , wherein said glycosylation site is the HCN1 site (SEQ ID NO: 10) of FIG. 12.  
     
     
         10 . The method of  claim 6 , wherein the antibody or antigen-binding fragment thereof is engineered to contain a glycosylation site is an antibody or antigen-binding fragment thereof having the binding specificity of the hLL2 antibody.  
     
     
         11 . The method of  claim 1 , wherein said glycosylation is located at a N-linked glycosylation site.  
     
     
         12 . The method of  claim 10 , wherein said expression vector comprises an amplifiable dihydrofolate reductase (dhfr) gene.  
     
     
         13 . The method of  claim 12 , wherein said expression vector is pdHL2.  
     
     
         14 . The method of  claim 13 , wherein said cell is a SP2/0 myeloma cell.  
     
     
         15 . The method of  claim 1 , wherein said antibody or fragment thereof is encoded by a DNA molecule comprising a DNA sequence comprising an engineered glycosylation site in the DNA sequence encoding the CH1 region or the constant light chain region.