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Abstract
Circulating HER2 extracellular domain (HER2 ECD) levels were proposed as a surrogate for HER2 tissue expression to monitor breast cancer patients for early relapse or responses to standard or HER2-targeted therapies, such as the monoclonal antibody (mAb) trastuzumab. Currently, available commercial ELISA assays for HER2 ECD rely on antibodies recognizing undisclosed or unknown epitopes. In this work, two ELISA assays employing MGR2 and MGR3 epitope-specific mAbs for HER2 ECD were developed and validated, showing good assay precision and linearity of the dose-response signal within the dynamic range of 0.19–12.50 ng mL−1 and detection limits of 0.76 and 0.75 ng mL−1 for the MGR2 and MGR3 assays, respectively. The developed assay showed a good agreement with two widely used commercial kits for HER2 ECD quantification in serum samples from breast cancer patients. A complete characterization of mAb-HER2 ECD interaction was performed by means of surface plasmon resonance using trastuzumab as control for both epitope mapping and kinetics analysis. The epitopes recognized by the two mAbs showed no overlap with trastuzumab, which was confirmed by trastuzumab interference analysis in serum samples. The method showed to be a practical approach to determine HER2 ECD with a high degree of sensitivity, reliability and recovery in samples containing mAbs-based therapies.
Highlights
HER2 is a trans-membrane tyrosine kinase receptor, member of the epidermal growth factor receptor (EGFR) family
Serum HER2 ECD is currently suggested in the follow-up and monitoring of patients with metastatic BC (MBC), whose initial serum HER2 value is above 15 ng mL−1 19, as high serum HER2 levels in MBC patients correlate with poor prognosis and decreased response to both conventional and anti-HER2 targeted anticancer therapies
In the present study we describe the development of sensitive enzyme-linked immunosorbent assays (ELISA), based on two epitope-specific monoclonal antibodies, namely MGR2 and MGR3, which were capable of correctly quantifying HER2 ECD in presence of trastuzumab
Summary
HER2 is a trans-membrane tyrosine kinase receptor, member of the epidermal growth factor receptor (EGFR) family. HER2 status of patients with BC and their eligibility for trastuzumab therapy have been determined using tissue-based tests such as immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH)[16] These histological techniques requiring invasive biopsies are indicated to assess HER2 status in the primary tumor but cannot be used in the follow-up to monitor the disease and the response to treatment[17,18]. In the present study we describe the development of sensitive enzyme-linked immunosorbent assays (ELISA), based on two epitope-specific monoclonal antibodies, namely MGR2 and MGR3, which were capable of correctly quantifying HER2 ECD in presence of trastuzumab. These mAbs were obtained through Balb/c mice immunization with HER2 overexpressing Calu[3] cells (lung adenocarcinoma)[20] and were reported to recognize HER2 ECD epitope I and epitope III, respectively[20,21], while trastuzumab recognises epitope IV14,22
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