Charrier-Savournin F, Vallaghe J, Mahlous I, Martinez S, Trinquet E
Cisbio Bioassays, Codolet, France. Institut de Genomique Fonctionnelle, Montpellier, France.
Miptec 2011, Basel, Switzerland
The development of promising new therapeutic strategies, such as monoclonal antibodies directed against Receptor Tyrosine kinase (RTK) has become a new approach to the treatment of cancers. The selection of potent and selective MAbs is the first critical step in the process, which runs from drug discovery to clinical trial phases. Here we present an innovative, universal and simple assay which enables the screening and the characterization of antibodies targeting cell-surface receptors. This method is based on the recently launched Tag-lite platform, which combines HTRF detection with the fluorescent labeling of receptors using the SNAP-Tag technology. On living cells, the binding of specific antibodies to a receptor of interest is detected through a time-resolved energy transfer occurring between an HTRF donor-labeled receptor and a secondary anti-species antibody labeled with an HTRF acceptor. This assay was successfully applied to the monitoring of antibody binding to a large set of RTK. By performing competition experiments with natural RTK ligands, our approach determines whether the previously selected antibodies were directed against the orthosteric binding site. Moreover, it enables complete antibody characterization by determining its affinity (KD) and pharmacokinetic (Koff) constants.