Antibodies having modified carbohydrate content and methods of preparation and use

Abstract

This invention provides a method of altering the affinity of an antibody for the antigen to which it is directed which comprises introducing into the variable region of the antibody a carbohydrate recognition site under conditions such that a carbohydrate binds to the site and thus attaches to the antibody. This invention also provides a method of modifying the carbohydrate content of an antibody which comprises deleting from a constant region of the antibody a carbohydrate recognition site which naturally occurs in such constant region of such antibody. Antibodies, e.g., monoclonal antibodies and human monoclonal antibodies, diagnostic test kits, DNA encoding antibodies, therapeutic agents, and methods for detecting the presence of a substance in a sample, and for recovering and purifying a substance from a sample are also provided.

What is claimed is:  
     
         1 . A method of altering the affinity of an antibody for the antigen to which it is directed which comprises introducing into the variable region of the antibody a carbohydrate recognition site under conditions such that a carbohydrate binds to the site and thus attaches to the antibody.  
     
     
         2 . A method of  claim 1 , wherein the antibody is a monoclonal antibody  
     
     
         3 . A method of  claim 1 , wherein the antibody is a human antibody.  
     
     
         4 . A method of  claim 1 , wherein the carbohydrate recognition site is introduced into the second hypervariable region of the antibody.  
     
     
         5 . A method of  claim 1 , wherein the carbohydrate recognition site is introduced into a variable region of the antibody which does not normally include a carbohydrate recognition site.  
     
     
         6 . A method of  claim 1 , wherein the carbohydrate recognition site is introduced into the variable region of the antibody by site-directed mutagenesis of DNA encoding the variable region of the antibody.  
     
     
         7 . A method of producing an antibody which may be more readily recovered or purified which comprises introducing into the variable region of the antibody a carbohydrate recognition site under conditions such that a carbohydrate binds to the site and thus attaches to the antibody.  
     
     
         8 . A method of  claim 7 , wherein the antibody is a monoclonal antibody.  
     
     
         9 . A method of  claim 7 , wherein the antibody is a human antibody.  
     
     
         10 . A method of  claim 7 , wherein the carbohydrate recognition site is introduced into the second hypervariable region of the antibody.  
     
     
         11 . A method of  claim 7 , wherein the carbohydrate recognition site is introduced into a variable region of the antibody which does not normally include a carbohydrate recognition site.  
     
     
         12 . A method of  claim 5 , wherein the carbohydrate recognition site is introduced into the variable region of the antibody by site directed mutagenesis of DNA encoding the variable region of the antibody.  
     
     
         13 . An antibody which does not occur in nature and which comprises a carbohydrate recognition site genetically engineered into a variable region of the antibody which does not naturally include a carbohydrate recognition site in such variable region.  
     
     
         14 . A monoclonal antibody of  claim 13 .  
     
     
         15 . A human antibody of  claim 13 .  
     
     
         16 . An antibody of  claim 13 , characterized by enhanced affinity for the antigen to which it is directed.  
     
     
         17 . An antibody of  claim 13  labeled with a detectable marker.  
     
     
         18 . An antibody of  claim 17 , wherein the detectable marker is a chromophore, fluorophore or radioactive moiety.  
     
     
         19 . A solid support to which an antibody of  claim 16  is bound.  
     
     
         20 . An altered antibody prepared by the method of  claim 1 .  
     
     
         21 . An antibody of  claim 13  further comprising carbohydrate attached to the carbohydrate recognition site genetically engineered into the variable region of the antibody.  
     
     
         22 . An antibody of  claim 13 , wherein the variable region is the second hypervariable region of the antibody.  
     
     
         23 . DNA encoding the antibody of  claim 21 .  
     
     
         24 . A method of modifying the carbohydrate content of an antibody which comprises deleting from a constant region of the antibody a carbohydrate recognition site which naturally occurs in such constant region of such antibody.  
     
     
         25 . A method of  claim 24 , wherein the antibody is a monoclonal antibody.  
     
     
         26 . A method of  claim 24 , wherein the antibody is a human antibody.  
     
     
         27 . A method of  claim 24 , wherein the deletion of the carbohydrate recognition site from the constant region of the antibody is effected by site-directed mutagenesis of DNA encoding such constant region of the antibody.  
     
     
         28 . A method of modifying the carbohydrate content of an antibody which comprises adding to a constant region of the antibody a carbohydrate recognition site which does not naturally occur in such constant region of such antibody.  
     
     
         29 . A method of  claim 28 , wherein the addition of the carbohydrate recognition site to the antibody is effected by site-directed mutagenesis of DNA encoding such constant region of the antibody.  
     
     
         30 . A method of modifying the biological effector function of an antibody which comprises modifying the carbohydrate content of the antibody using the method of  claim 24 .  
     
     
         31 . A human antibody which does not occur in nature and which is characterized by the absence of a carbohydrate recognition site in a constant region of the antibody which naturally includes such a carbohydrate recognition site in such constant region.  
     
     
         32 . A human monoclonal antibody of  claim 31 .  
     
     
         33 . A human antibody which does not occur in nature and which is characterized by the presence of a carbohydrate recognition site in a constant region of the antibody which does not naturally include such carbohydrate recognition site in such constant region.  
     
     
         34 . A human monoclonal antibody of  claim 33 .  
     
     
         35 . A human antibody of  claim 33  having carbohydrate attached to such carbohydrate recognition site.  
     
     
         36 . A labeled antibody of  claim 35 , wherein the label is bound to such carbohydrate.  
     
     
         37 . A therapeutic agent comprising the antibody of  claim 35  and a therapeutic ligand, the ligand being bound to such carbohydrate attached to such antibody.  
     
     
         38 . A modified antibody prepared by the method of  claim 24 .  
     
     
         39 . A modified antibody prepared by the method of  claim 28 .  
     
     
         40 . DNA encoding the antibody of  claim 31 .  
     
     
         41 . DNA encoding the antibody of  claim 33 .  
     
     
         42 . A more sensitive method for detecting the presence of a substance in a sample which comprises contacting the sample with an antibody of  claim 17  directed to the substance, under conditions such that substance in the sample forms a detectable complex with the antibody, and detecting the complex and thus the substance.  
     
     
         43 . A method of  claim 42 , wherein the antibody is bound to a solid support.  
     
     
         44 . A quantitative method of  claim 42 , wherein the amount of complex formed is determined and thereby the amount of the substance present in the sample.  
     
     
         45 . A method of recovering a substance from a sample containing the substance which comprises contacting the sample with an antibody of  claim 13  directed to the substance under conditions such that substance in the sample forms a complex with the antibody and recovering the substance from the resulting complex.  
     
     
         46 . A chromatographic method of  claim 45 , wherein the antibody is bound to a solid support.  
     
     
         47 . A purification method for a substance which comprises recovering the substance using the method of  claim 45  from a sample under conditions such that the substance is recovered in purified form.  
     
     
         48 . A diagnostic test kit comprising the antibody of  claim 13 .  
     
     
         49 . A diagnostic test kit comprising the antibody of claim  17 .