Receptor tyrosine kinase biomarkers

Abstract

The present invention relates to the use of receptor tyrosine kinase (RTK) oligomers as markers of RTK activation and signalling. Methods are described based upon determining the presence of RTK oligomers and/or determining the nanometre spatial separation between RTK molecules assembled as RTK oligomers at the cell surface. Such methods are directed to the monitoring of RTK activation in cells and the detection of mutations in RTKs. Methods are also described for determining prognosis for subjects having diseases characterised by aberrant RTK activation and for selecting subjects for treatment with RTK inhibitors.

1 . A method of monitoring receptor tyrosine kinase (RTK) activation in a cell, the method comprising the steps of:
 (i) imaging RTK molecules at the cell surface; and   (ii) determining the presence of one or more RTK oligomers wherein the RTK oligomers comprise more than two RTK monomers,   wherein the one or more RTK oligomers is/are indicative of RTK activation in said cell.   
     
     
         2 - 41 . (canceled) 
     
     
         42 . A method comprising:
 measuring a pharmacological response to a therapeutic agent comprising:
 (i) detecting the presence of one or more receptor tyrosine kinase (RTK) oligomers in one or more cells in a sample obtained from a subject by contacting the one or more cells with a fluorophore-conjugated RTK ligand or a fluorophore-conjugated RTK-specific affibody, and detecting binding between RTK monomers and the fluorophore-conjugated RTK ligand or the fluorophore-conjugated RTK-specific affibody, wherein RTK is EGFR/ErbB1, ErbB2, ErbB3 or ErbB4, and wherein each RTK oligomer comprises more than two RTK monomers, 
 (ii) measuring the nanometer (nm) spatial separation between RTK monomers assembled in any of the one or more RTK oligomers in the absence of the therapeutic agent, 
 (iii) contacting the cells with the therapeutic agent, and 
 (iv) measuring the nm spatial separation between RTK monomers assembled in any of the one or more RTK oligomers in the presence of the therapeutic agent. 
   
     
     
         43 . A method comprising:
 measuring a pharmacological response to a therapeutic agent comprising:
 (i) imaging one or more receptor tyrosine kinase (RTK) oligomers at the surface of a cell in the absence and in the presence of the therapeutic agent, wherein the RTK oligomers comprise more than two RTK monomers, and wherein the presence of one or more RTK oligomers is indicative of RTK activation in a cell, and 
 (ii) indicating the therapeutic agent as an RTK inhibitor if it inhibits aberrant activation of the imaged RTK molecules in the cell. 
   
     
     
         44 . The method of  claim 42 , wherein the nanometre (nm) spatial separation between RTK monomers assembled in any of the one or more RTK oligomers is indicative of RTK activation. 
     
     
         45 . The method of  claim 42 , wherein the presence of one or more RTK oligomers indicates that the subject is at increased risk of disease progression. 
     
     
         46 . The method of  claim 45 , wherein the disease progression is cancer progression. 
     
     
         47 . The method of  claim 42 , wherein the therapeutic agent is an RTK inhibitor. 
     
     
         48 . The method of  claim 43 , wherein the RTK inhibitor is selected from an EGFR family inhibitor, a VEGFR inhibitor, and a PDGF inhibitor. 
     
     
         49 . The method of  claim 42 , wherein the therapeutic agent is an EGFR inhibitor. 
     
     
         50 . The method of  claim 49 , wherein the EGFR inhibitor is selected from Gefitinib (Iressa), Erlotinib (Tarceva), Lapatinib, Canertinib, Cetuximab (Erbitux) and Panitumumab (Vectibix). 
     
     
         51 . The method of  claim 42 , further comprising administering the therapeutic agent to the subject. 
     
     
         52 . The method of  claim 42 , wherein the RTK monomers are imaged using a high-resolution imaging method. 
     
     
         53 . The method of  claim 52 , wherein the high-resolution imaging method is capable of determining intermolecular distances in the range from 5-80 nm. 
     
     
         54 . The method of  claim 52 , wherein the high-resolution imaging method is Fluorophore Localisation Imaging with Photobleaching (FLImP). 
     
     
         55 . The method of  claim 42 , wherein the RTK oligomers are selected from tetramers, hexamers, or higher-order oligomers consisting of at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 RTK monomers. 
     
     
         56 . The method of  claim 42 , wherein the RTK oligomers are tetramers. 
     
     
         57 . The method of  claim 43 , wherein the RTK is selected from EGFR, ErbB2, ErbB3 and ErbB4. 
     
     
         58 . The method of  claim 42 , wherein the RTK is EGFR. 
     
     
         59 . The method of  claim 43 , wherein the RTK is EGFR. 
     
     
         60 . The method of  claim 59 , wherein the nanometre spatial separation of EGFR monomers is measured using fluorophore-conjugated EGF ligands.