Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action

Assays for activated HER1 and HER2 receptors in FFPE and cell lysate formats were developed using VeraTag？ technology, which requires the proximity of an antibody pair for light-dependent release of a fluorescently labeled tag, followed by capillary electrophoresis-based quantitation.Ligand-dependent and independent HER1-HER2 heterodimer levels measured by lysate and FFPE VeraTag？ assays trended with HER1 and HER2 expression levels in tumor cell lines, which was confirmed by co-immunoprecipitation. The formation of EGF-dependent HER1-HER2 heterodimers were inhibited by the HER2-targeted monoclonal antibody 2C4 and stabilized by the HER1 tyrosine kinase inhibitor (TKI) erlotinib. EGF-dependent HER1 and HER2 phosphorylation was inhibited by lapatinib and erlotinib. Further, we observed that dominant receptor signaling patterns may switch between HER1-HER1 and HER1-HER2, depending on drug mechanism of action and relative levels of HER receptors. In FFPE breast tumors that expressed both HER1 and HER2, HER1-HER2 heterodimers were detected in 25 to 50% of tumors, depending on detection method. The levels of activated phospho-HER1-HER2 heterodimers correlated with HER1 or HER2 levels in an analysis of 43 HER2-positive breast tumors.VeraTag？ lysate assays can be used as a tool for understanding the mechanism of action of targeted HER-family inhibitors in the preclinical setting, while VeraTag？ FFPE assays of activated HER receptors combined with total HER2 measurements (HERmark？) in tumor samples may provide a more accurate prediction of clinical response to both HER1 and HER2 targeted therapies.Both the epidermal growth factor receptor (EGFR/HER1) and HER2 are members of the ErbB family of the type I receptor tyrosine kinases, which also includes HER3 and HER4. These homologous receptors are comprised of an extracellular binding domain (ECD), a transmembrane domain, and an intracellular tyrosine kinase (TK) domain. Binding of ligand to the ECD induces structural reorganiz