KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements
Autor: | Jonas Stenberg, Marc Vanhove, Diana Spiegelberg, Pascale Richalet |
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Rok vydání: | 2021 |
Předmět: |
0301 basic medicine
Biophysics Computational biology law.invention Flow cytometry 03 medical and health sciences 0302 clinical medicine law medicine Panitumumab Receptor End point medicine.diagnostic_test Live-cells measurements Chemistry General Medicine Biofysik Receptor–ligand kinetics LigandTracer 030104 developmental biology Affinity Binding kinetics 030220 oncology & carcinogenesis Recombinant DNA Pertuzumab medicine.drug |
Zdroj: | European Biophysics Journal. 50:979-991 |
ISSN: | 1432-1017 0175-7571 |
Popis: | 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 KD 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. |
Databáze: | OpenAIRE |
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