Methods for increasing the diversity of monoclonal antibodies produced against an antigen

Abstract

The present invention relates to methods for increasing the diversity of monoclonal antibodies produced against an antigen. The methods of the invention utilize immunization of a murine host defective in one or more enzymes involved in a post-translational modification of a polypeptide or a modification of a lipid, wherein said modification is exposed on a cell surface. The invention also relates to monoclonal antibodies produced by these methods and which are not produced when a normal mouse is immunized with the same antigen. The invention further relates to compositions comprising these monoclonal antibodies, as well as to such monoclonal antibodies bound or conjugated to a toxin, a detectable marker or to a solid support.

1 . A method of producing an antibody library that comprises an antibody that binds to an epitope on a molecule of interest, wherein the epitope is not present naturally in a wild-type host, the method comprising the steps of:
 (a) immunizing a knockout host with an antigen that comprises the epitope present on said molecule of interest in a manner sufficient for said knockout host to develop antibodies against said epitope, wherein when the wild-type host is immunized with the antigen it does not produce an antibody that binds to the epitope, wherein said knockout host comprises a knockout mutation in an enzyme involved in post-translational glycosylation of a;   (b) isolating splenocytes or B cells from said immunized host;   (c) extracting ribonucleic acid (RNA) from the isolated splenocytes or B cells; and   (d) generating an antibody library from the extracted RNA.   
     
     
         2 . The method of  claim 1 , wherein the library is a phage display library. 
     
     
         3 . The method of  claim 1 , wherein the library is a yeast display library. 
     
     
         4 . The method of  claim 1  further comprising the step of amplifying the RNA by polymerase chain reaction (PCR). 
     
     
         5 . The method of  claim 1  wherein the B cells are isolated from blood, bone marrow, or lymph node. 
     
     
         6 . The method of  claim 1  wherein the splenocytes or B cells obtained in step (b) are immortalized. 
     
     
         7 . The method of  claim 6 , wherein the splenocytes or B cells are immortalized by viral infection or transduction. 
     
     
         8 . The method of  claim 7  wherein the isolated splenocytes or B cells are fused with other cells. 
     
     
         9 . The method of  claim 1 , wherein the host is a non-human mammal. 
     
     
         10 . The method of  claim 1  wherein the host is a genetically modified non-human animal comprising one or more human immunoglobulin sequences that encode an antibody gene. 
     
     
         11 . A method of producing RNA that encodes for an antibody that binds to an epitope that is not present naturally in a wild-type host, the method comprising the steps of:
 (a) immunizing a knockout host with an antigen that comprises the epitope in a manner sufficient for said knockout host to develop antibodies against said epitope, wherein when the wild-type host is immunized with the antigen it does not produce an antibody that binds to the epitope, wherein said knockout host comprises a knockout mutation in an enzyme involved in post-translational glycosylation of a polypeptide; and   (b) amplifying RNA isolated from B cells of said immunized host by PCR.   
     
     
         12 . The method of  claim 11  wherein the B cells are from blood, bone marrow, or lymph node. 
     
     
         13 . The method of  claim 11  further comprising the step of generating an antibody library from the isolated RNA. 
     
     
         14 . The method of  claim 13 , wherein the library is a phage display library. 
     
     
         15 . The method of  claim 13 , wherein the library is a yeast display library.