Specific binding agents

Abstract

A reshaped human antibody or reshaped human antibody fragment having specificity for human polymorphic epithelial mucin (PEM) is produced by transferring the complementarity determining regions (CDRs) from a murine anti-HMFG hybridoma cell line HMFG1 into a human antibody variable region framework. The reshaped molecule can be used in the treatment or diagnosis of cancer.

1 . A synthetic specific binding agent having specificity for human polymorphic epithelial mucin (PEM), conferred by the presence of one or more of the amino acid sequences: 
 i) Ala Tyr Trp Ile Glu    ii) Glu Ile Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Lys Gly    iii) Ser Tyr Asp Phe Ala Trp Phe Ala Tyr    iv) Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr Leu Ala    v) Trp Ala Ser Thr Arg Glu Ser    vi) Gln Gln Tyr Tyr Arg Tyr Pro Arg Thr    
     
     
         2  A reshaped human antibody, or a reshaped human antibody fragment, having specificity for human polymorphic epithelial mucin (PEM) conferred by the presence of one or more of the amino acid sequences: 
 i) Ala Tyr Trp Ile Glu  
 ii) Glu Ile Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Lys Gly  
 iii) Ser Tyr Asp Phe Ala Trp Phe Ala Tyr  
 iv) Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr Leu Ala  
 v) Trp Ala Ser Thr Arg Glu Ser  
 vi) Gln Gln Tyr Tyr Arg Tyr Pro Arg Thr  
 
     
     
         3 . A reshaped human antibody or reshaped human antibody fragment according to  claim 2 , having at least one heavy-chain variable region incorporating the following CDRs: 
 CDR1: Ala Tyr Trp Ile Glu    CDR2: Glu Ile Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Lys Gly    CDR3: Ser Tyr Asp Phe Ala Trp Phe Ala Tyr    
     
     
         4 . A reshaped human antibody or reshaped human antibody fragment according to  claim 2 , having at least one light-chain variable region incorporating the following CDRs: 
 CDR1: Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr Leu Ala    CDR2: Trp Ala Ser Thr Arg Glu Ser    CDR3: Gln Gln Tyr Tyr Arg Tyr Pro Arg Thr    
     
     
         5 . A reshaped human antibody or reshaped human antibody fragment according to  claim 2  and having at least one heavy-chain variable region according to  claim 3  and at least one light-chain variable region according to  claim 4 .  
     
     
         6 . A reshaped human antibody or reshaped human antibody fragment according to  claim 2 , incorporating at least one heavy-chain variable region comprising the entire amino acid sequence depicted in FIG. 12 of the accompanying drawings.  
     
     
         7 . A reshaped human antibody or reshaped human antibody fragment according to  claim 2 , incorporating at least one light-chain variable region comprising the entire amino acid sequence depicted in FIG. 13 of the accompanying drawings.  
     
     
         8 . A synthetic specific binding agent, reshaped human antibody or reshaped human antibody fragment, according to any one of the preceding claims, wherein the PEM is human milk fat globule (HMFG).  
     
     
         9 . A synthetic specific binding agent, reshaped human antibody or reshaped human antibody fragment, having specificity equivalent to that of the gamma-1, kappa anti-HMFG monoclonal antibody “HMFG1”.  
     
     
         10 . A stable host cell line producing a synthetic specific binding agent, reshaped human antibody or reshaped human antibody fragment according to any one of  claims 1  to  9 , resulting from incorporation in the cell line of a foreign gene encoding the synthetic specific binding agent, reshaped human antibody or reshaped human antibody fragment.  
     
     
         11 . A stable host cell line according to  claim 10 , wherein the foreign gene includes one or more of the nucleotide sequences: 
 i) GCC TAC TGG ATA GAG    ii) GAG ATT TTA CCT GGA AGT AAT AAT TCT AGA TAC AAT GAG AAG TTC AAG GGC    iii) TCC TAC GAC TTT GCC TGG TTT GCT TAC    iv) AAG TCC AGT CAG AGC CTT TTA TAT AGT AGC AAT CAA AAG ATC TAC TTG GCC    v) TGG GCA TCC ACT AGG GAA TCT    vi) CAG CAA TAT TAT AGA TAT CCT CGG ACG    
     
     
         12 . A stable host cell line according to  claim 10 , wherein the foreign gene includes the entire nucleotide sequence depicted in FIG. 12 of the accompanying drawings.  
     
     
         13 . A stable host cell line according to  claim 10 , wherein the foreign gene includes the entire nucleotide sequence depicted in FIG. 13 of the accompanying drawings.  
     
     
         14 . A stable host cell line according to  claim 10 , wherein the foreign gene encodes: 
 a) at least one of the amino acid sequences: 
 i) Ala Tyr Trp Ile Glu  
 ii) Glu Ile Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Lys Gly  
 iii) Ser Tyr Asp Phe Ala Trp Phe Ala Tyr  
 iv) Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr Leu Ala  
 v) Trp Ala Ser Thr Arg Glu Ser  
 vi) Gln Gln Tyr Tyr Arg Tyr Pro Arg Thr  
 and b) a protein framework that enables the encoded amino acid sequence when expressed to function as a CDR having specificity for PEM.  
   
     
     
         15 . A stable host cell line according to  claim 10 , wherein the foreign gene encodes the entire amino acid sequence depicted in FIG. 12 of the accompanying drawings.  
     
     
         16 . A stable host cell line according to  claim 10 , wherein the foreign gene encodes the entire amino acid sequence depicted in FIG. 13 of the accompanying drawings.  
     
     
         17 . Plasmid pSVgpt-HuVHHMFG1-HuIgG1.  
     
     
         18 . Plasmid pSVneo-HuVkHMFG1-HuCk.  
     
     
         19 . Use of plasmid according to  claim 17  or  claim 18  in the production of a synthetic specific binding agent, reshaped human antibody or reshaped human antibody fragment.  
     
     
         20 .  E.coli  NCTC 12411.  
     
     
         21 .  E.coli  NCTC 12412.  
     
     
         22 . A DNA sequence encoding a reshaped human antibody heavy-chain variable region having specificity for HMFG, as contained in  E.coli  NCTC 12411.  
     
     
         23 . A DNA sequence encoding a reshaped human antibody light-chain variable region having specificity for HMFG, as contained in  E.coli  NCTC 12412.  
     
     
         24 . A reshaped human antibody heavy-chain variable region having specificity for HMFG, producible by means of the expression vector contained in  E.coli  NCTC 12411.  
     
     
         25 . A reshaped human antibody light-chain variable region having specificity for HMFG, producible by means of the expression vector contained in  E.coli  NCTC 12412.  
     
     
         26 . A reshaped human antibody or reshaped human antibody fragment, comprising at least one variable region according to  claim 24  or  claim 25 .  
     
     
         27 . A synthetic specific binding agent, reshaped human antibody or reshaped human antibody fragment, according to any one of  claims 1  to  9  or  claim 26 , linked to or incorporating an agent capable of retarding or terminating the growth of cancerous cells, or linked to an agent capable of being detected while inside the human body.  
     
     
         28 . An injectable composition comprising a synthetic specific binding agent, reshaped human antibody or reshaped human antibody fragment, according to  claim 27 , in a pharmaceutically acceptable carrier.  
     
     
         29 . Use of a synthetic specific binding agent, reshaped human antibody or reshaped human antibody fragment, according to any one of  claims 1  to  9  or  claim 26 , for the manufacture of a medicament for therapeutic application in the relief of cancer in humans, or for the manufacture of a diagnostic composition for in-vivo diagnostic application in humans.  
     
     
         30 . Use of a synthetic binding agent, reshaped human antibody or reshaped human antibody fragment, according to  claim 27 , in a method of human cancer therapy or imaging.