Modifided carboxypeptidase enzymes and their use

Abstract

The invention relates to improvements relating to cancer therapy based on the identification of a number of regions of CPG2 which contain epitopes which appear to be involved in the production of a host immune response and which may be modified to alter the immunogenicity in patients. Production of fusions of CPG2 with an antibody, where the CPG2 protein has been tagged provides a CPG2 protein which has reduced immunogenicity. By using partially glycosylated enzyme obtainable by P. pastoris expression, the efficacy of antibody-CPG2 fusions is enhanced.

1 - 15 . (Canceled)  
     
     
         16 . An isolated carboxypeptidease enzyme, CPG2, in which an immunogenic region is modified to reduce immunogenicity to a mammalian immune system whilst retaining CPG2 activity, wherein the immunogenic region is selected from the group consisting of:  
       
         
           
                 
                 
                 
               
                     
                 
                   (i) 
                   KIDGRGGK (SEQ ID NO.1) comprising residues 98-105 of SEQ ID NO.7; 
                     
                 
                     
                 
                   (ii) 
                   KEYGVRD (SEQ ID NO.2) comprising residues 157-163 of SEQ ID NO.7; 
                 
                     
                 
                   (iii) 
                   YGVRD (SEQ ID NO.6) comprising residues 159-163 of SEQ ID NO.7; 
                 
                     
                 
                   (iv) 
                   KLADY (SEQ ID NO.3) comprising residues 191-195 of SEQ ID NO.7; 
                 
                     
                 
                   (v) 
                   GAGK (SEQ ID NO.4) comprising residues 412 to the C-terminal residue 415 of SEQ ID NO.7; 
                 
                     
                 
                   (vi) 
                   AG comprising residues 413-414 of SEQ ID NO.7; 
                 
                   and 
                 
                     
                 
                   (vii) 
                   EGGKKLVDK (SEQ ID NO.5) comprising residues 331-339 of SEQ ID NO.7. 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         17 . An isolated or purified  Pseudomonas  carboxypeptidase CPG2 enzyme wherein the C-terminus of the enzyme comprises an extension selected from the group consisting of a histidine tag, a myc tag and a myc-his tag.  
     
     
         18 . An isolated carboxypeptidease enzyme, CPG2, in which an immunogenic region is modified to reduce immunogenicity to a mammalian immune system whilst retaining CPG2 activity, wherein the immunogenic region is selected from the group consisting of: 
 (i) KIDGRGGK (SEQ ID NO.1) comprising residues 98-105 of SEQ ID NO.7;    (ii) KEYGVRD (SEQ ID NO.2) comprising residues 157-163 of SEQ ID NO.7;    (iii) YGVRD (SEQ ID NO.6) comprising residues 159-163 of SEQ ID NO. 7;    (iv) KLADY (SEQ ID NO.3) comprising residues 191-195 of SEQ ID NO.7;    (v) GAGK (SEQ ID NO.4) comprising residues 412 to the C-terminal residue 415 of SEQ ID NO.7;    (vi) AG comprising residues 413-414 of SEQ ID NO.7; and    (vii) EGGKKLVDK (SEQ ID NO.5) comprising residues 331-339 of SEQ ID NO.7;    wherein the C-terminus of the enzyme comprises an extension selected from the group consisting of a histidine tag, a myc tag and a myc-his tag.    
     
     
         19 . The carboxypeptidase enzyme of  claim 16  wherein said enzyme is fused to an antibody other than an anti-CEA antibody.  
     
     
         20 . The carboxypeptidase enzyme of  claim 17  wherein said enzyme is fused to an antibody other than an anti-CEA antibody.  
     
     
         21 . The carboxypeptidase enzyme of  claim 18  wherein said enzyme is fused to an antibody other than an anti-CEA antibody.  
     
     
         22 . A method of preparing a fusion protein comprising a carboxypeptidase CPG2 enzyme of  claim 17  and an antibody other than a CEA-antibody, comprising expressing a DNA sequence encoding said fusion operably linked to a promoter in a  Pichia pastoris  host cell, and recovering said fusion protein therefrom.  
     
     
         23 . A method of preparing a fusion protein comprising a carboxypeptidase CPG2 enzyme of  claim 18  and an antibody other than a CEA-antibody, comprising expressing a DNA sequence encoding said fusion operably linked to a promoter in a  Pichia pastoris  host cell, and recovering said fusion protein therefrom.  
     
     
         24 . A kit comprising a first component which is a prodrug which can be converted to a cytotoxic drug by a carboxypeptidase of  claim 16  and, as a second component, said carboxypeptidase.  
     
     
         25 . A kit comprising a first component which is a prodrug which can be converted to a cytotoxic drug by a carboxypeptidase of  claim 17  and, as a second component, said carboxypeptidase.  
     
     
         26 . A kit comprising a first component which is a prodrug which can be converted to a cytotoxic drug by a carboxypeptidase of  claim 18  and, as a second component, said carboxypeptidase.  
     
     
         27 . A kit comprising a first component which is a prodrug which can be converted to a cytotoxic drug by a carboxypeptidase of  claim 19  and, as a second component, said carboxypeptidase.  
     
     
         28 . A kit comprising a first component which is a prodrug which can be converted to a cytotoxic drug by a carboxypeptidase of  claim 20  and, as a second component, said carboxypeptidase.  
     
     
         29 . A kit comprising a first component which is a prodrug which can be converted to a cytotoxic drug by a carboxypeptidase of  claim 21  and, as a second component, said carboxypeptidase.