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 increasing the diversity of monoclonal antibodies that bind to a molecule of interest comprising the steps of: 
 a) immunizing a murine host with an antigen that comprises an epitope present on said molecule in a manner sufficient for said host to develop antibodies against said antigen, 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, said modification being exposed on a cell surface or present on a secreted molecule; and wherein said molecule of interest is not present naturally in a murine host.    b) isolating the spleenocytes from said immunized host and fusing said splenocytes with immortalized cells to form a hybridoma; and    c) assaying said hybridoma to determine it produces a monoclonal antibody that bind to said molecule.    
   
   
       2 . The method according to  claim 1 , wherein said host has a knockout mutation in an enzyme selected from a glycosyltransferase, a sulfotransferase, a protease involved in proteolytic processing, or an acetyltransferase.  
   
   
       3 . The method according to  claim 2 , wherein said murine host is selected from a mouse having a knockout mutation in mannosyl (α1,6-)-glycoprotein β-1,6-N-acetyl-glucosaminyltransferase V (MGAT5; GNT-V), a mouse having a knockout mutation in core 2 β1-6N-acetylglucosaminyltransferase (C2 GlcNAcT), or a mouse having a knockout mutation in β(1,4)-galactosyltransferase I (β4GalT-I).  
   
   
       4 . The method according to  claim 2 , wherein said antigen is selected from a leukocyte cell surface antigen, a viral envelope antigen, or a cancer cell membrane antigen.  
   
   
       5 . The method according to  claim 4 , wherein said antigen is selected from PSGL-1, a membrane preparation from a human breast cancer tumor or HIV viral envelope gp120.  
   
   
       6 . In a method of producing monoclonal antibodies that bind to a molecule of interest comprising the steps of immunizing a murine host with an antigen that comprises an epitope present on said molecule; allowing the host to develop antibodies to said antigen; isolating the spleenocytes of said host; fusing said spleenocytes with immortalized cells to form a hybridoma; and assaying individual hybridoma cells to determine if said hybridoma cell produces a monoclonal antibody that binds to said molecule, wherein said molecule of interest is not present naturally in a murine host, the improvement comprising utilizing a murine host comprising a knockout mutation in an enzyme involved in a post-translational modification of a polypeptide or a modification of a lipid, said modification being exposed on a cell surface or present on a secreted molecule.  
   
   
       7 . The method according to  claim 6 , wherein said host has a knockout mutation in an enzyme selected from a glycosyltransferase, a sulfotransferase, a protease involved in proteolytic processing, or an acetyltransferase.  
   
   
       8 . The method according to  claim 7 , wherein said murine host is selected from a mouse having a knockout mutation in mannosyl (α1,6-)-glycoprotein β-1,6-N-acetyl-glucosaminyltransferase V (MGAT5; GNT-V), a mouse having a knockout mutation in core 2 β1-6N-acetylglucosaminyltransferase (C2 GlcNAcT), or a mouse having a knockout mutation in β(1,4)-galactosyltransferase I (β4GaIT-I).  
   
   
       9 . The method according to  claim 7 , wherein said antigen is selected from a leukocyte cell surface antigen, a viral envelope antigen, or a cancer cell membrane antigen  
   
   
       10 . The method according to  claim 9 , wherein said antigen is selected from PSGL-1, a membrane preparation from a human breast cancer tumor or HIV viral envelope gp120.  
   
   
       11 . A hybridoma comprising: 
 a) a B cell from a murine host that has been immunized with an antigen that comprises an epitope present on a molecule of interest, 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, said modification being exposed on a cell surface or present on a secreted molecule and wherein said molecule is not present naturally in a murine host, fused to    b) an immortalized cell, wherein said hybridoma produces a monoclonal antibody characterized in that it binds to said molecule of interest; and is not produced by a hybridoma comprising: 
 i) a B cell from a murine host that is normal in said enzyme and that is immunized with said antigen, fused to  
 ii) an immortalized cell.  
   
   
   
       12 . A method of producing a monoclonal antibody comprising the steps of: 
 a) growing the hybridoma according to  claim 11  under conditions that promote the expression of said monoclonal antibody; and    b) isolating said monoclonal antibody from media in which said hybridoma is grown.    
   
   
       13 . A method of producing a monoclonal antibody comprising the steps of: 
 a) identifying a hybridoma according to  claim 11;     b) isolating from said hybridoma DNA encoding said monoclonal antibody;    c) optionally modifying said DNA so as to encode for a modified or derivatized antibody selected from a humanized antibody, a chimeric antibody, a single chain antibody or an immunoreactive fragment of an antibody;    d) inserting said DNA into an expression vector;    e) transfecting a host compatible with said expression vector; and    f) growing said transfected host under conditions that cause the expression of the antibody encoded by said DNA, wherein said modified or derivatized antibody binds to said molecule of interest; and is not produced by a hybridoma comprising: 
 i) a B cell from a murine host normal in said enzyme that is immunized with said antigen, fused to  
 ii) an immortalized cell.  
   
   
   
       14 . An isolated monoclonal antibody, modified monoclonal antibody, or derivatized monoclonal antibody produced by the method of  claim 12  or  13 .  
   
   
       15 . A composition comprising: 
 a) the isolated monoclonal antibody, modified monoclonal antibody or derivatized monoclonal according to  claim 14  in an amount effective to detectably reduce or eliminate the activity of said molecule of interest in a patient; and    b) a pharmaceutically acceptable carrier.    
   
   
       16 . A method of inhibiting the activity or function of a molecule of interest in a patient or a biological sample comprising the step of contacting said patient or biological sample with a composition according to  claim 15 .  
   
   
       17 . An isolated monoclonal antibody according to  claim 14  conjugated or covalently bound to a toxin, a detectable moiety or a solid support.  
   
   
       18 . The monoclonal antibody according to  claim 17 , wherein said detectable moiety is selected from a fluorescent moiety, a radioisotope or an imaging agent.  
   
   
       19 . A composition comprising: 
 a) an isolated monoclonal antibody according to  claim 14;     b) a liposome; and    c) a substance selected from a nucleic acid sequence, a toxin or a drug, wherein said composition is formulated such that the monoclonal antibody selectively targets the delivery of said substance when administered to an animal.    
   
   
       20 . A method of delivering a functional gene to the cell of an animal comprising the step of administering the composition according to  claim 19  to said animal.  
   
   
       21 . A method of knocking out a functional gene in a cell of an animal comprising the step of administering the composition according to  claim 19  to said animal.  
   
   
       22 . A method of delivering a toxin or a drug to a cell in an animal comprising the step of administering the composition according to  claim 19  to said animal.