Specific binding proteins and uses thereof

Abstract

The invention relates to specific binding members, particularly antibodies and active fragments thereof, which recognize an aberrant post-translationally modified, particularly an aberrant glycosylated form of the EGFR. The binding members, particularly antibodies and fragments thereof, of the invention do not bind to EGFR on normal cells in the absence of amplification of the wild-type gene and are capable of binding the de2-7 EGFR at an epitope which is distinct from the junctional peptide. Antibodies of this type are exemplified by the novel antibody 806 whose VH and VL sequences are illustrated as SEQ ID NOs: 2 and 4 and chimeric antibodies thereof as exemplified by ch806.

1 . An antibody which recognizes an EGFR epitope which does not demonstrate any amino acid sequence alterations or substitutions of normal EGFR and which is found in tumorigenic, hyperproliferative or abnormal cells and not detectable in normal cells, wherein said epitope is located within the region comprising amino acid residues 273-501 of normal EGFR. 
     
     
         2 . An antibody which recognizes an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells and not detectable in normal cells, wherein the epitope is enhanced or evident upon aberrant expression, wherein said specific binding member is capable of binding the de2-7 EGFR at an epitope distinct from the junctional peptide and which does not bind to EGFR on normal cells in the absence of aberrant expression, and wherein said epitope is located within the region comprising amino acid residues 273-501 of normal EGFR. 
     
     
         3 . An antibody which recognizes an EGFR epitope which does not demonstrate any amino acid sequence alterations or substitutions from normal EGFR and which is found in tumorigenic, hyperproliferative or abnormal cells and not detectable in normal cells wherein said antibody comprises the polypeptide sequence set out in either one or both of SEQ ID NO:2 or SEQ ID NO:4. 
     
     
         4 . An antibody which recognizes an EGFR epitope which is found in tumorigenic, hyperproliferative or abnormal cells and not detectable in normal cells, wherein the epitope is enhanced or evident upon aberrant expression, wherein said antibody is capable of binding the de2-7 EGFR at an epitope distinct from the junctional peptide and does not bind to EGFR on normal cells in the absence of amplification of the normal wild-type gene, and wherein said antibody comprises the polypeptide sequence set out in either one or both of SEQ ID NO:2 or SEQ ID NO:4. 
     
     
         5 . The antibody of  claims 3  or  4 , wherein the polypeptide sequence is as set out in SEQ ID NO:2. 
     
     
         6 . The antibody of  claims 3  or  4 , wherein the polypeptide sequence is as set out in SEQ ID NO:4. 
     
     
         7 . An antibody according to  claim 5 , wherein said antibody comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out as any one or more of residues 26-35A, 49-64 or 93-102 of SEQ ID NO:2. 
     
     
         8 . An antibody according to  claim 7 , wherein said polypeptide binding domain is substantially as set out as residues 26-35A of SEQ ID NO:2. 
     
     
         9 . An antibody according to  claim 7 , wherein said polypeptide binding domain is substantially as set out as residues 49-64 of SEQ ID NO:2. 
     
     
         10 . An antibody according to  claim 7 , wherein said polypeptide binding domain is substantially as set out as residues 93-102 of SEQ ID NO:2. 
     
     
         11 . An antibody according to  claim 6 , wherein said antibody comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out as any one or more of residues 24-34, 50-56 or 89-97 of SEQ ID NO:4. 
     
     
         12 . An antibody according to  claim 11 , wherein said polypeptide binding domain is substantially as set out as residues 24-34 of SEQ ID NO:4. 
     
     
         13 . An antibody according to  claim 11 , wherein said polypeptide binding domain is substantially as set out as residues 50-56 of SEQ ID NO:4. 
     
     
         14 . An antibody according to  claim 11 , wherein said polypeptide binding domain is substantially as set out as residues 89-97 of SEQ ID NO:4. 
     
     
         15 . An antibody according to any one of  claims 1  to  14 , wherein said binding domains are carried by a human IgG1 antibody framework. 
     
     
         16 . An antibody according to any one of  claims 1  to  14 , wherein said binding domains are carried by a human IgG1, human kappa antibody framework. 
     
     
         17 . An antibody capable of binding the de2-7 EGFR at an epitope distinct from the junctional peptide and which does not bind to EGFR on normal cells in the absence of amplification of the normal wild-type gene, wherein said antibody comprises the polypeptide sequence set out in SEQ ID NO:4. 
     
     
         18 . An antibody according to any one of the preceding claims in the form of an antibody F(ab′) 2  or scFv fragment. 
     
     
         18 . An antibody according to any one of  claims 1  to  17 , which carries a detectable or functional label. 
     
     
         19 . An antibody according to  claim 18 , wherein said label is a covalently attached drug. 
     
     
         20 . An antibody according to  claim 18 , wherein said label is a radiolabel. 
     
     
         21 . An isolated nucleic acid which comprises a sequence encoding an antibody as defined in any one of  claims 1  to  17 . 
     
     
         22 . A method of preparing an antibody as defined in any one of  claims 1  to  17  which comprises expressing the nucleic acid of  claim 21  under conditions to bring about expression of said binding member, and recovering the binding member. 
     
     
         23 . A specific binding member or antibody according to any one of  claims 1  to  17  for use in a method of treatment or diagnosis of cancer in the human or animal body. 
     
     
         24 . A method of preparing a specific binding member capable of binding a tumor antigen, which method comprises:
 a) providing a starting repertoire of nucleic acids encoding a VH domain which lack a CDR3 encoding region;   b) combining said repertoire with a donor nucleic acid encoding an amino acid sequence substantially as set out as in any one or more of residues 26 to 35A, 49 to 64 or 93 to 102 of SEQ ID NO:2 such that said donor nucleic acid is inserted into the missing CDR3 region, so as to provide a product repertoire of nucleic acids encoding a VH domain;   c) expressing the nucleic acids of said product repertoire; and   d) selecting a specific binding member which has a maximum tumor:blood localization ratio in a test animal of >1:1 and optionally at said ratio, a non-tumor bearing organ to blood ratio of <1:1; and   e) recovering said binding member or the nucleic acid encoding it.   
     
     
         25 . A method of treatment of a tumor in a human patient which comprises administering to said patient an effective amount of an antibody as defined in any one of  claims 1  to  17 . 
     
     
         26 . A kit for the diagnosis of a tumor in which EGFR is aberrantly expressed or EGFR is expressed in the form of a truncated protein, said kit comprising a binding member or antibody of any one of  claims 1  to  17 , optionally with reagents and/or instructions for use. 
     
     
         27 . A pharmaceutical composition comprising the antibody of any of  claims 1 - 17 , and optionally, a pharmaceutically acceptable vehicle, carrier or diluent. 
     
     
         28 . A kit for the treatment of a tumor in a human patient, comprising a pharmaceutical dosage form of the pharmaceutical composition of  claim 27 , and a separate pharmaceutical dosage form of the tyrosine kinase inhibitor AG1478. 
     
     
         29 . A kit for the treatment of a tumor in a human patient, comprising a pharmaceutical dosage form of the pharmaceutical composition of  claim 27 , and a separate pharmaceutical dosage form of the anti-EGFR antibody mAb 528. 
     
     
         30 . A unicellular host transformed with a recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes the antibody of  claims 1 - 4 , or a fragment thereof, selected from the group consisting of:
 (A) the DNA sequence of  FIG. 13  (SEQ ID NO: 1);   (B) the DNA sequence of  FIG. 15  (SEQ ID NO:3);   (C) the DNA sequence of  FIG. 13  (SEQ ID NO: 1) and the DNA sequence of  FIG. 15  (SEQ ID NO:3);   (D) the DNA sequence of  FIG. 13  (SEQ ID NO: 1) with a constant IgG1 sequence as set out in SEQ ID NO:8 and the DNA sequence of  FIG. 15  (SEQ ID NO:3) with a constant kappa sequence as set out in SEQ ID NO:7;   (E) DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions; and   (F) DNA sequences that code on expression for an amino acid sequence encoded by any of the foregoing DNA sequences;   wherein said DNA sequence is operatively linked to an expression control sequence.   
     
     
         31 . The unicellular host of  claim 30 , wherein the unicellular host is selected from the group consisting of  E. coli, Pseudomonas, Bacillus, Streptomyces , yeasts, CHO, YB/20, NSO, SP2/0, R1.1, B-W, L-M, COS1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, and human cells in tissue culture. 
     
     
         32 . A method for detecting the presence of amplified EGFR, de2-7EGFR or EGFR with high mannose glycosylation wherein said EGFR is measured by:
 A. contacting a biological sample from a mammal in which the presence of amplified EGFR, de2-7EGFR or EGFR with high mannose glycosylation is suspected with an antibody capable of specifically binding to said EGFR under conditions that allow binding of said EGFR to said antibody to occur; and   B. detecting whether binding has occurred between said EGFR from said sample and the antibody;   wherein the detection of binding indicates that presence or activity of said EGFR in said sample.   
     
     
         33 . A method for detecting cancer in mammals comprising detecting the presence or activity of an EGFR according to the method of  claim 32 , wherein detection of the presence of the EGFR indicates the existence of a tumor or cancer in said mammal. 
     
     
         34 . A method of preventing and/or treating cancer in mammals, comprising administering to a mammal a therapeutically effective amount of the pharmaceutical composition of  claim 27  or the kit of either one of  claims 28  or  29 . 
     
     
         35 . A method for the treatment of brain-resident cancers that produce aberrantly expressed EGFR in mammals, comprising administering to a mammal a therapeutically effective amount of the pharmaceutical composition of  claim 27  or the kit of either one of  claims 28  or  29 . 
     
     
         36 . The method of  claim 34 , wherein said brain-resident cancers are selected from glioblastomas, medulloblastomas, meningiomas, neoplastic astrocytomas and neoplastic arteriovenous malformations. 
     
     
         37 . The method of  claims 35  and  36 , wherein said pharmaceutical composition is administered systemically. 
     
     
         38 . A method for the treatment of malignant neural tumors in mammals, comprising administering to a mammal a therapeutically effective amount of the pharmaceutical composition of  claims 26 - 27  or the kit of  claim 28 . 
     
     
         39 . An antibody comprising a variable heavy chain with the polypeptide sequence as set out in SEQ ID NO:2 and a human IgG1 constant region. 
     
     
         40 . An antibody comprising a variable light chain with the polypeptide sequence as set out in SEQ ID NO:4 and a human kappa constant region. 
     
     
         41 . An antibody comprising a variable heavy chain with the polypeptide sequence as set out in SEQ ID NO:2. 
     
     
         42 . An antibody comprising a variable light chain with the polypeptide sequence as set out in SEQ ID NO:4. 
     
     
         43 . An antibody, wherein said antibody comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out as any one or more of residues 26-35A, 49-64 or 93-102 of SEQ ID NO:2. 
     
     
         44 . An antibody, wherein said antibody comprises a polypeptide binding domain comprising an amino acid sequence substantially as set out as any one or more of residues 24-34, 50-56 or 89-97 of SEQ ID NO:4.