Recombinant peptides derived from the Mc3 anti-BA46 antibody, methods of use thereof, and methods of humanizing antibody peptides

Abstract

The present invention provides recombinant peptides that specifically and selectively bind to the human milk fat globule (HMFG) antigen, BA46. In particular, the present invention provides recombinant variants of the Mc3 antibody, including humanized versions of Mc3. The variant Mc3 peptides are particularly useful for diagnostic, prognostic, and therapeutic applications in the field of breast cancer. The present invention also provides methods for the humanization of antibodies such as murine monoclonal antibodies. The novel humanization methods are applied to the production of humanized Mc3 antibodies and it is shown that these humanized antibodies retain the ability to engage in high affinity binding to their cognate antigen. Such humanization enables the use of these antibodies for immunodiagnostic and immunotherapeutic applications in humans.

1 . A recombinant Mc3 antibody which binds to BA46 antigen of the human milk fat globule (HMFG), said antibody comprising at least one modified variable region, said modified variable region selected from the group consisting of: 
 (i) a modified heavy chain variable region having an amino acid sequence substantially similar to that of murine Mc3 in FIG. 12 in which at least one but fewer than about 30 of the amino acid residues of murine Mc3 have been substituted; and    (ii) a modified light chain variable region having an amino acid sequence substantially similar to that of murine Mc3 in FIG. 13 in which at least one but fewer than about 30 of the amino acid residues of murine Mc3 have been substituted; and    (iii) a derivative of one of said modified variable regions in which one or more residues of the variable region that are not required for binding to the antigen have been deleted or in which one or more of the residues labelled (CDR) in FIG. 12 or  13  have been modified without disrupting antigen binding.    
     
     
         2 . A recombinant murine Mc3 antibody of  claim 1 , wherein at least one of said substituted amino acids is replaced with the corresponding amino acid from the appropriate human consensus sequence of FIG. 12 or  13 , for a heavy or light chain variable region, respectively.  
     
     
         3 . A recombinant Mc3 antibody of  claim 1  wherein said antibody comprises a heavy chain variable region and a light chain variable region.  
     
     
         4 . A recombinant Mc3 antibody of  claim 3  wherein both variable regions are modified variable regions, and wherein the antibody further comprises an antibody constant region or other effector agent.  
     
     
         5 . A recombinant Mc3 antibody of  claim 4  wherein the antibody comprises a constant region that is a human antibody constant region.  
     
     
         6 . A recombinant Mc3 antibody of  claim 1  wherein at least about five of the amino acid residues in one of said modified variable regions have been replaced with corresponding amino acids from the appropriate human consensus sequence of FIG. 12 or  13 , for a heavy or light chain variable region, respectively.  
     
     
         7 . A recombinant Mc3 antibody of  claim 1  comprising a modified heavy chain variable region in which at least about half of the residues listed as humanized or humanized (BR) in FIG. 12 have been replaced with corresponding residues from the human consensus sequence of FIG. 12.  
     
     
         8 . A recombinant Mc3 antibody of  claim 1  comprising a modified light chain variable region in which at least about half of the residues listed as humanized or humanized (BR) in FIG. 13 have been replaced with corresponding residues from the human consensus sequence of FIG. 13.  
     
     
         9 . A recombinant Mc3 antibody of  claim 5  comprising 
 a modified heavy chain variable region in which at least about 90% of the residues listed as humanized or humanized (BR) in FIG. 12 have been replaced with corresponding residues from the human consensus sequence of FIG. 12; and  
 a modified light chain variable region in which at least about 90% of the residues listed as humanized or humanized (BR) in FIG. 13 have been replaced with corresponding residues from the human consensus sequence of FIG. 13.  
 
     
     
         10 . A recombinant Mc3 antibody of  claim 9  in which all of the residues listed as humanized or humanized (BR) have been replaced with corresponding residues from the human consensus sequences of FIGS.  12  or  13 , for the heavy and light chains respectively.  
     
     
         11 . A pharmaceutical composition comprising a recombinant Mc3 antibody of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         12 . A nucleic acid sequence encoding a modified variable region of  claim 1 .  
     
     
         13 . A nucleic acid sequence of  claim 12  comprising the coding region of a modified variable region as shown in FIG. 18 or  19 .  
     
     
         14 . An in vitro method of detecting the presence of an HMFG antigen or binding fragment thereof, comprising 
 obtaining a biological sample suspected of comprising the antigen or a fragment thereof;    adding a recombinant Mc3 antibody of  claim 1  under conditions effective to form an antibody-antigen complex; and    detecting the presence of said antibody-antigen complex.    
     
     
         15 . A method of diagnosing the presence of an HMFG antigen or binding fragment thereof in a subject, comprising 
 administering to the subject a recombinant Mc3 antibody of  claim 1  under conditions effective to deliver it to an area of the subject's body suspected of containing an HMFG antigen or a binding fragment thereof to form an antibody-antigen complex; and    detecting the presence of said antibody-antigen complex.    
     
     
         16 . A method of delivering an agent to a target site that contains an HMFG antigen comprising 
 binding said agent to a recombinant Mc3 antibody of  claim 1  at a position other than the antigen binding site to create an agent-antibody complex; and    introducing the agent-antibody complex to the environment of said target site under conditions suitable for binding of an antibody to its cognate antigen.    
     
     
         17 . A method of  claim 16 , wherein the target site is within the body of a human subject and introducing the agent-antibody complex comprises administering the complex to said subject.  
     
     
         18 . A method of humanizing a non-human antibody comprising replacing one or more framework amino acid residues in a variable region of said antibody with corresponding framework amino acids from a human variable region wherein important non-human framework residues, as defined by the buried-residue-modification technique, are retained in their original form.  
     
     
         19 . A method of humanizing a non-human antibody comprising replacing one or more framework amino acid residues in a variable region of said antibody with corresponding framework amino acids from a human variable region consensus sequence wherein important non-human framework residues, as defined by the buried-residue-modification technique, are retained in their original form.  
     
     
         20 . A method of  claim 19  wherein both the heavy and the light chain variable regions of said antibody are humanized.