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.

What is claimed is: 
     
         1 . A method of increasing the diversity of monoclonal antibodies that bind to a molecule of interest comprising the steps of:
 (a) immunizing a host with an antigen that comprises an epitope present on said molecule in a manner sufficient for said host to develop antibodies against said epitope, wherein said host comprises a knockout mutation in an enzyme involved in a post-translational modification of a polypeptide or a modification of a lipid; and   (b) isolating splenocytes or B cells from said immunized host.   
     
     
         2 . The method of  claim 1  further comprising the step of extracting ribonucleic acid (RNA) from isolated splenocytes or B cells. 
     
     
         3 . The method of  claim 2  further comprising the step of generating an antibody library from the extracted RNA. 
     
     
         4 . The method of  claim 3 , wherein the library is a phage display library. 
     
     
         5 . The method of  claim 3 , wherein the library is a yeast display library. 
     
     
         6 . The method of  claim 2  further comprising the step of amplifying the RNA by polymerase chain reaction (PCR). 
     
     
         7 . The method of  claim 1  wherein the B cells are isolated from blood, bone marrow, or lymph node. 
     
     
         8 . The method of  claim 1  further comprising the step of immortalizing splenocytes or B cells isolated from said immunized host. 
     
     
         9 . The method of  claim 8 , wherein the step of immortalizing is by viral infection or transduction. 
     
     
         10 . The method of  claim 8 , further comprising the step of extracting RNA from the immortalized cells. 
     
     
         11 . The method of  claim 10 , further comprising the step of amplifying the extracted RNA by PCR. 
     
     
         12 . The method of  claim 10 , further comprising the step of generating an antibody library from the extracted RNA. 
     
     
         13 . The method of  claim 1  further comprising the step of fusing isolated splenocytes or B cells with other cells. 
     
     
         14 . The method of  claim 1 , wherein the host is a non-human mammal 
     
     
         15 . 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. 
     
     
         16 . A method of increasing the diversity of monoclonal antibodies that bind to a molecule of interest comprising the steps of:
 (a) immunizing a host with an antigen that comprises an epitope present on said molecule in a manner sufficient for said host to develop antibodies against said epitope, wherein said host comprises a knockout mutation in an enzyme involved in a post-translational modification of a polypeptide or a modification of a lipid; and   (b) amplifying RNA isolated from B cells of said immunized host by PCR.   
     
     
         17 . The method of  claim 16  wherein the B cells are from blood, bone marrow, or lymph node. 
     
     
         18 . The method of  claim 16  further comprising the step of generating an antibody library from the isolated RNA. 
     
     
         19 . The method of  claim 18 , wherein the library is a phage display library. 
     
     
         20 . The method of  claim 18 , wherein the library is a yeast display library.