Journal
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS
Volume 50, Issue 7, Pages 979-991Publisher
SPRINGER
DOI: 10.1007/s00249-021-01560-2
Keywords
LigandTracer; Flow cytometry; Affinity; Binding kinetics; Live-cells measurements
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The design of next-generation therapeutics faces new challenges, and the study characterizes the interaction of five different antibody therapeutics with their target receptors using LigandTracer. This method can be performed on live cells without the need for a recombinant source of the receptor and provides information on binding kinetics and affinity, complementing flow cytometry experiments.
Design of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point K-D determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.
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