Antibodies as a cancer diagnostic

Abstract

Method and kits are provided for the detection and diagnosis of metastatic disease. More particularly, the methods and kits employ compounds that can detect EphA2, a specific epithelial cell tyrosine kinase that is overexpressed in metastatic tumor cells. In one embodiment the compound is an antibody capable of binding to an epitope of EphA2.

1 . A method for detecting the presence of metastatic cells in a cell population comprising the steps of 
 lysing at least a portion of the cell population,    incubating the lysed cells , with a reagent capable of specific binding to an epitope of EphA2 to allow, antibody binding to said epitope, and    detecting compound-epitope binding.    
   
   
       2 . The method of  claim 1  wherein the reagent is an antibody.  
   
   
       3 . The method of  claim 2  wherein the epitope of EphA2 is and intracellular epitope of EphA2.  
   
   
       4 . The method of  claim 3  wherein the antibody is produced by hybridoma cell line D7.  
   
   
       5 . The method of  claim 2  wherein the antibody is labeled with a detectable label, and the detecting step includes detecting the label.  
   
   
       6 . The method of  claim 5  wherein the antibody is labeled wvitlh a fluorescent label and the detecting step comprises detecting the fluorescent label.  
   
   
       7 . The method of  claim 5  wherein the antibody is labeled with a radioactive label and the detecting step comprises detecting the radioactive label.  
   
   
       8 . The method of  claim 1  wherein the cell population comprises cells from a breast or prostate tissue biopsy.  
   
   
       9 . The method of  claim 1  wherein the cell population is harvested from a body fluid selected from the group consisting of blood, plasma, spinal fluid, saliva, and urine.  
   
   
       10 . The method of  claim 9  wherein the detecting step includes a diagnostic method selected from the group consisting of ELISA assays and flow cytometry.  
   
   
       11 . The method of  claim 1  wherein the incubating and detecting steps comprise western blotting methodology.  
   
   
       12 . The method of  claim 11  further comprising the steps of providing a second antibody having phosphotyrosine specificity, and western blotting with the second antibody.  
   
   
       13 . The method of  claim 1  wherein the metastatic cells are selected from the group consisting of breast, prostate, lung, and colon cancers.  
   
   
       14 . An antibody which specifically binds to an intracellular epitope of EphA2.  
   
   
       15 . The antibody of  claim 14  bound to a detectable label.  
   
   
       16 . The antibody of  claim 14  which is a monoclonal antibody.  
   
   
       17 . A method for detecting the presence of metastatic cells in a cell population comprising the steps of: 
 incubating the cells with a reagent capable of specific binding to a compound associated with EphA2 expression, and    detecting reagent-compound binding.    
   
   
       18 . The method of  claim 17  wherein the reagent is an antibody.  
   
   
       19 . The method of  claim 17  wherein the compound is selected from the group consisting of EphA2, a fragment of EphA2, DNA coding for the EphA2 protein, and RNA coding for the EphA2 protein.  
   
   
       20 . The method of  claim 17  further comprising the step of fixing the cells on a slide, and the detecting step comprises immunofluorescence staining.  
   
   
       21 . A kit for detecting the presence of metastatic cells in a cell population comprising 
 an antibody capable of specific binding to an epitope of EphA2, and    means for detecting antibody-epitope binding.    
   
   
       22 . The kit of  claim 21  wherein the means for detecting antibody-epitope binding is a label bound to the antibody.  
   
   
       23 . The kit of  claim 21  further comprising an antibody having phosphotyrosine specificity.  
   
   
       24 . A method for detecting the presence of metastatic or potentially metastatic cells in a cell population comprising: 
 lysing at least a portion of the cell population;    incubating the lysed cells with a reagent capable of specific binding to EphA2 to allow binding of the reagent to EphA2; and    detecting reagent-EphA2 binding, wherein reagent-EphA2 binding is indicative of the presence of metastatic or potentially metastatic cells in the cell population.    
   
   
       25 . The method of  claim 24  wherein the reagent comprises an antibody, and wherein detecting reagent-EphA2 binding comprises detecting antibody-EphA2 binding.  
   
   
       26 . The method of  claim 25  wherein the antibody binds to an intracellular epitope of EphA2.  
   
   
       27 . The method of  claim 25  wherein the antibody is produced by hybridoma cell line D7.  
   
   
       28 . The method of  claim 25  wherein the antibody is labeled with a detectable label, and wherein detecting reagent-EphA2 binding comprises detecting the label.  
   
   
       29 . The method of  claim 28  wherein the antibody comprises at least one of a fluorescent label, a chemiluminescent label, a bioluminescent label, an enzymatic label, a chromogenic label and a radiolabel, wherein detecting reagent-EphA2 binding comprises detecting at least one detectable label.  
   
   
       30 . The method of  claim 24  wherein the cell population comprises cells selected from the group consisting of breast cells, kidney cells, prostate cells, lung cells and colon cells.  
   
   
       31 . The method of  claim 24  wherein the cell population comprises epithelial cells.  
   
   
       32 . The method of  claim 24  wherein the cell population comprises cells selected from the group consisting of breast cancer cells, kidney cancer cells, prostate cancer cells, lung cancer cells and colon cancer cells.  
   
   
       33 . The method of  claim 24  wherein the cell population comprises epithelial cancer cells.  
   
   
       34 . The method of  claim 24  wherein the cell population comprises metastatic or potentially metastatic cancer cells.  
   
   
       35 . The method of  claim 34  wherein the metastatic or potentially metastatic cancer cells comprise cells selected from the group consisting of breast cancer cells, kidney cancer cells, prostate cancer cells, lung cancer cells, and colon cancer cells.  
   
   
       36 . The method of  claim 34  wherein the metastatic or potentially metastatic cancer cells comprise epithelial cancer cells.  
   
   
       37 . The method of  claim 24  wherein the cell population comprises cells from a tissue biopsy.  
   
   
       38 . The method of  claim 37  wherein the tissue comprises breast tissue or prostate tissue.  
   
   
       39 . The method of  claim 24  wherein the cell population comprises cells from a body fluid.  
   
   
       40 . The method of  claim 39  wherein the body fluid is selected from the group consisting of blood, plasma, spinal fluid, saliva, and urine.  
   
   
       41 . The method of  claim 24  wherein detecting reagent-EphA2 binding comprises utilizing a diagnostic method selected from the group consisting of an ELISA assay, a Western blot, and flow cytometry.  
   
   
       42 . The method of  claim 24  wherein detecting reagent-EphA2 binding comprises utilizing a Western blot; the method further comprising Western blotting with a second antibody having phosphotyrosine specificity.  
   
   
       43 . A method for detecting the presence of metastatic or potentially metastatic cells in a cell population comprising: 
 incubating at least a portion of the cell population with a reagent capable of binding to EphA2 to allow binding of the reagent to EphA2; and    detecting reagent-EphA2 binding, wherein reagent-EphA2 binding is indicative of the presence of metastatic or potentially metastatic cells in the cell population.    
   
   
       44 . The method of  claim 43  wherein the reagent comprises an antibody and wherein detecting reagent-EphA2 binding comprises detecting antibody-EphA2 binding.  
   
   
       45 . The method of  claim 44  wherein the antibody binds to an intracellular epitope of EphA2.  
   
   
       46 . The method of  claim 44  wherein the antibody is produced by hybridoma, cell line D7.  
   
   
       47 . The method of  claim 44  wherein the antibody binds to an extracellular epitope of EphA2.  
   
   
       48 . The method of claim of  claim 44  wherein antibody-EphA2 binding yields a bound complex comprising a whole cell.  
   
   
       49 . The method of  claim 48  wherein detecting antibody-EphA2 binding comprises subjecting the bound complex to immunohistochemical staining.  
   
   
       50 . The method of  claim 44  wherein the antibody is produced by hybridoma cell line B2D6.  
   
   
       51 . The method of  claim 44  wherein the bound antibody comprises a detectable label; and wherein detecting antibody-EphA2 binding comprises detecting the label.  
   
   
       52 . The method of  claim 44  wherein the bound antibody comprises at least one of a fluorescent label, a chemiluminescent label, a bioluminescent label, an enzymatic label, a chromogenic label and a radiolabel; and wherein detecting antibody-Eph2 binding comprises detecting, at least one detectable label.  
   
   
       53 . The method of  claim 43  wherein the cell population comprises cells selected from the group consisting of breast cells, kidney cells, prostate cells, lung cells and colon cells.  
   
   
       54 . The method of  claim 43  wherein the cell population comprises epithelial cells.  
   
   
       55 . The method of  claim 43  wherein the cell population comprises cells selected from the group consisting of breast cancer cells, kidney cancer cells, prostate cancer cells, lung cancer cells and colon cancer cells.  
   
   
       56 . The method of  claim 43  wherein the cell population comprises epithelial cancer cells.  
   
   
       57 . The method of  claim 43  wherein the cell population comprises metastatic or potentially metastatic cancer cells.  
   
   
       58 . The method of  claim 57  wherein the metastatic or potentially metastatic cells comprise cells selected from the group consisting of breast cancer cells, kidney cancer cells, prostate cancer cells, lung cancer cells, and colon cancer cells.  
   
   
       59 . The method of  claim 43  wherein the metastatic or potentially metastatic cells comprise epithelial cancer cells.  
   
   
       60 . The method of  claim 43  wherein the cell population comprises cells from a tissue biopsy.  
   
   
       61 . The method of  claim 60  wherein the tissue comprises breast tissue or prostate tissue.  
   
   
       62 . The method of  claim 43  wherein the cell population comprises cells from a body fluid.  
   
   
       63 . The method of  claim 62  wherein the body fluid is selected from the group consisting of blood, plasma, spinal fluid, saliva, and urine.  
   
   
       64 . The method of  claim 43  wherein detecting reagent-EphA2 binding comprises utilizing a diagnostic method selected from the group consisting of and ELISA assay, a Western blot, anal flow cytometry.  
   
   
       65 . The method of  claim 43  wherein detecting reagent-EphA2 binding comprises utilizing a Western blot; the method further comprising Western blotting with a second antibody having phosphotyrosine specificity.  
   
   
       66 . A method for detecting the presence of metastatic or potentially metastatic cells in a cell population comprising: 
 lysing at least a portion of the cell population;    incubating the lysed cells with a reagent capable of specific binding to nucleic acid comprising RNA or DNA encoding at least a portion of an EphA2 protein to allow binding of the reagent to the nucleic acid; and    detecting reagent-nucleic acid binding, wherein reagent-nucleic acid binding is indicative of the presence of metastatic or potentially metastatic cells in the cell population.    
   
   
       67 . The method of  claim 66  wherein the nucleic acid comprises RNA.  
   
   
       68 . A method for detecting the presence of cancer cells in a selected cell population comprising: 
 assaying at least a portion of the selected cell population for at least one of 
 a change in EphA2 expression level;  
 a change in EphA2 localization pattern; and  
 a change in EphA2 phosphorylation content  
   as compared to the EphA2 expression level, localization pattern and phosphorylation content in an analogous normal cell population;    wherein the change is indicative of the presence of a cancer cell in the selected cell population.    
   
   
       69 . The method of  claim 68  wherein a change in EphA2 expression level, localization pattern or phosphorylation content is indicative of the presence of metastatic cancer cells in the cell population.  
   
   
       70 . The method of  claim 68  wherein a change in EphA2 expression level is indicative of the presence of nonmetastatic cancer cells in the cell population.  
   
   
       71 . The method of  claim 68  wherein assaying the cell population comprises incubating at least a portion of the selected cell population with a reagent capable of binding to EphA2 to allow binding of the reagent to EphA2; and detecting reagent-EphA2 binding.  
   
   
       72 . The method of  claim 71  wherein the reagent is an antibody.  
   
   
       73 . The method of  claim 72  wherein the antibody is produced by hybridoma D7 or B2D6.  
   
   
       74 . A method for determining the disease stage in a cell population comprising cancer cells, the method comprising: 
 assaying at least a portion of the cell population for at least one of 
 EphA2 expression level;  
 EphA2 localization; and  
 EphA2 phosphorylation content; and  
   determining the disease stage of the cancer cells.    
   
   
       75 . The method of  claim 74  wherein assaying the cell population comprises incubating at least a portion of the cancer cell population with a reagent capable of binding to EphA2 to allow binding of the reagent to EphA2; and detecting reagent-EphA2 binding.  
   
   
       76 . The method of  claim 75  wherein the reagent is an antibody.  
   
   
       77 . The method of  claim 76  wherein the antibody is produced by hybridoma D7 or B2D6.  
   
   
       78 . An isolated antibody which specifically binds to an intracellular epitope of EphA2.  
   
   
       79 . The isolated antibody of  claim 78  which is a monoclonal antibody.