Methods for diagnosis of cancer using erbB-3

Abstract

A DNA fragment distinct from the epidermal growth factor receptor (EGFR) and erbB-2 genes was detected by reduced stringency hybridization of v-erbB to normal genomic human DNk Characterization of the cloned DNA fragment mapped the region of v-erbB homology to three exons with closest homology of 64% and 67% to a contiguous region within the tyrosine kinase domains of the EGFR and erbB-2 proteins, respectively. cDNA cloning revealed a predicted 148 kd transmembrane polypeptide with structural features identifying it as a member of the erbB family, prompting designation of the new gene as erbB-3. It was mapped to human chromosome 12q11-13 and was shown to be expressed as a 6.2 kb transcript in a variety of normal tissues of epithelial origin. Markedly elevated erbB-3 mRNA levels were demonstrated in certain human mammary tumor cell lines. These findings indicate that increased erbB-3 expression, as in the case of EGFR and erbB-2, plays a role in some human malignancies. Using erbB-3 specific antibodies (polyclonal or monoclonal), the erbB-3 protein was identified as a 180 kDa glycoprotein, gp180 erbB-3 . The intrinsic catalytic function of gp180 erbB-3 was uncovered by its ability to autophosphorylate in vitro. Ligand-dependent signaling of its cytoplasmic domain was established employing transfectants which express a chimeric EGFR/erbB-3 protein, gp180 EGFR/erbB-3 . EGF induced tyrosine phosphorylation of the chimera and promoted soft agar colony formation of such transfectants. These findings, combined with the detection of constitutive tyrosine phosphorylation of gp180 erbB-3 in 4 out of 12 human mammary tumor cell lines, implicate the activated erbB-3 product in the pathogenesis of some human malignancies. Thus, this invention also relates to a method for detecting a receptor ligand capable of either activating or down-regulating the receptor protein, as well as procedures for purifying the resultant ligand; a method of screening potential ligand analogs for their ability to activate the receptor protein; and procedures for targeting a therapeutic drug to cells having a high level of the receptor protein.

What is claimed is:  
     
         1 . An isolated polypeptide having the amino acid sequence as given by SEQ ID NO: 4, or a unique portion thereof.  
     
     
         2 . The isolated polypeptide of  claim 1 , wherein the unique portion is selected from the group consisting of an amino acid sequence given by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.  
     
     
         3 . An isolated polypeptide having an amino acid sequence encoded by the DNA isolate consisting essentially of a nucleotide sequence that encodes an amino acid sequence differing in at least one amino acid from the amino acid sequence of human erbB-3, or a unique portion thereof, and having greater overall similarity to the amino acid sequence of human erbB-3 than to that of any other polypeptide.  
     
     
         4 . A purified antibody specific for a unique portion of SEQ ID NO: 4 or gp180 erbB-3 .  
     
     
         5 . The antibody of  claim 4 , wherein the unique portion is selected from the group consisting of an amino acid sequence given by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.  
     
     
         6 . The antibody of  claim 4 , wherein the antibody is conjugated to a therapeutic drug.  
     
     
         7 . The antibody of  claim 4 , wherein the antibody is labeled with a detectable moiety.  
     
     
         8 . The antibody of  claim 4 , wherein the antibody is bound to a solid support.  
     
     
         9 . A bioassay for determining the amount of erbB-3 mRNA in a biological sample comprising the steps of: 
 a) contacting the biological sample with a nucleic acid isolate consisting essentially of a nucleotide sequence that encodes erbB-3 or a unique portion thereof under conditions such that a hybrid molecule can be formed; and    b) determining the amount of hybrid molecule present, the amount of hybrid molecule indicating the amount of erbB-3 mRNA in the sample.    
     
     
         10 . A bioassay for screening potential analogs of ligands of erbB-3 receptors for the ability to affect an activity mediated by the erbB-3 receptors, comprising the steps of: 
 a) contacting a molecule suspected of being a ligand with erbB-3 receptors produced by a cell that yields functional erbB-3 receptors;    b) determining-the amount of a biological activity mediated by the erbB-3 receptors; and    c) selecting those analogs which affect the biological activity mediated by the erbB-3 receptor.    
     
     
         11 . A bioassay for determining the amount of an erbB-3 antigen in a biological sample comprising the steps of: 
 a) contacting the sample with an antibody according to  claim 4  under conditions such that a specific complex of the antibody and the antigen can be formed; and    b) determining the amount of the antibody present as the complex, the amount of the complex indicating the amount of erbB-3 antigen in the biological sample.    
     
     
         12 . A method for targeting a therapeutic drug to cells having high levels of erbB-3 receptors, comprising the steps of: 
 a) conjugating an antibody according to according to  claim 4  or an active fragment thereof to the drug; and    b) administering the resulting conjugate to an individual with cells having high levels of erbB-3 receptors in an effective amount and by an effective route such that the antibody is able to bind to the receptor on the cells.    
     
     
         13 . A method for detecting the presence of an erbB-3 activating ligand in a source containing a potential erbB-3 ligand, comprising the steps of: 
 a) contacting a first sample of cells from a cell line that expresses erbB-3 protein with the source containing a potential erbB-3 ligand for a time and under conditions sufficient to allow erbB-3 ligand contained in the source to bind to erbB-3 protein to form a triggered sample, wherein the cell line expresses erbB-3 protein having low level intrinsic tyrosine phosphorylation;    b) contacting a second sample of cells from the cell line with a control medium for the time and under the conditions as given in step a) to form a control sample;    c) determining the level of erbB-3 activation in the triggered sample and in the control sample; and    d) comparing the level of erbB-3 activation in the triggered sample with the level of erbB-3 activation in the control sample, wherein an increase in activation in the triggered sample over the control sample indicates the presence of an erbB-3 activating ligand in the source containing a potential erbB-3 ligand.    
     
     
         14 . The method of  claim 13 , wherein the determining step comprises measuring the level of erbB-3 intrinsic tyrosine phosphorylation in the triggered sample and in the control sample, wherein an increase in the level of erbB-3 intrinsic tyrosine phosphorylation correlates with an increase in the level of erbB-3 activation.  
     
     
         15 . The method of  claim 13 , wherein the determining step comprises measuring the level of cell growth in the triggered sample and in the control sample, wherein an increase in the level of cell growth correlates with an increase in the level of erbB-3 activation.  
     
     
         16 . The method of  claim 13 , wherein the determining step comprises measuring the level of DNA synthesis for the cells in the triggered sample and in the control sample, wherein an increase in the level of DNA synthesis for the cells correlates with an increase in the level of erbB-3 activation.  
     
     
         17 . The method of  claim 13 , wherein erbB-3 is overexpressed by the cell line.  
     
     
         18 . The method of  claim 13 , further comprising the step of purifying the erbB-3 activating ligand.  
     
     
         19 . A method for detecting the presence of an erbB-3 blocking ligand in a source containing a potential erbB-3 ligand, comprising the steps of: 
 a) contacting a first sample of a cell line that expresses erbB-3 protein with the source containing a potential erbB-3 ligand for a time and under conditions sufficient to allow erbB-3 ligand contained in the source to bind to erbB-3 protein to form a blocked sample, wherein the cell line expresses erbB-3 protein having high level intrinsic tyrosine phosphorylation;    b) contacting a second sample of the cell line with a control medium for the time and under the conditions as given in step a) to form a control sample;    c) determining the level of erbB-3 activation in the blocked sample and in the control sample; and    d) comparing the level of erbB-3 activation in the blocked sample with the level of erbB-3 activation in the control sample, wherein a decrease in activation in the blocked sample over the control sample indicates the presence of an erbB-3 blocking ligand in the source containing a potential erbB-3 ligand.    
     
     
         20 . The method of  claim 19 , wherein the determining step comprises measuring the level of erbB-3 intrinsic tyrosine phosphorylation in the blocked sample and in the control sample, wherein an decrease in the level of erbB-3 intrinsic tyrosine phosphorylation correlates with an decrease in the level of erbB-3 activation.  
     
     
         21 . The method of  claim 19 , wherein the determining step comprises measuring the level of cell growth in the blocked sample and in the control sample, wherein an decrease in the level of cell growth correlates with an decrease in the level of erbB-3 activation.  
     
     
         22 . The method of  claim 19 , wherein the determining step comprises measuring the level of DNA synthesis for the cells in the blocked sample and in the control sample, wherein an decrease in the level of DNA synthesis for the cells correlates with an decrease in the level of erbB-3 activation.  
     
     
         23 . The method of  claim 19 , wherein erbB-3 is overexpressed by the cell line.  
     
     
         24 . The method of  claim 19 , further comprising the step of purifying the erbB-3 blocking ligand.  
     
     
         25 . A purified erbB-3 activating ligand.  
     
     
         26 . A purified erbB-3 blocking ligand.  
     
     
         27 . A method of decreasing a biochemical or biological activity mediated by the erbB-3 receptor, comprising blocking the binding of an erbB-3 activating ligand with the erbB-3 receptor.  
     
     
         28 . The method of  claim 27 , wherein the blocking is accomplished by an antibody reactive with the ligand binding domain of the erbB-3 receptor.  
     
     
         29 . The method of  claim 27 , wherein the blocking is accomplished by an erbB-3 blocking ligand.  
     
     
         30 . A method of promoting a biochemical or biological activity mediated by the erbB-3 receptor, comprising contacting an erbB-3 activating ligand with the erbB-3 receptor.  
     
     
         31 . A method of detecting the overexpression of erbB-3 in a sample from a subject, comprising detecting the amount of erbB-3 in the sample and comparing the amount in the sample to the amount in an equivalent sample having normal expression, the presence of erbB-3 in a greater amount indicating overexpression of erbB-3.  
     
     
         32 . The method of  claim 31 , further comprising correlating the overexpression of erbB-3 to a tumor.  
     
     
         33 . A method of detecting the activation of erbB-3 in a test sample from a subject, comprising detecting the presence of phosphorylation of erbB-3, the presence of phosphorylation of erbB-3 indicating the presence of erbB-3 activation in the sample.  
     
     
         34 . The method of  claim 33 , further comprising comparing the amount of erbB-3 phosphorylation in the test sample to the amount of erbB-3 phosphorylation in a sample from a normal subject and correlating an increase in phosphorylation in the test sample, with the presence of a neoplastic condition in the subject.