KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

Diana Spiegelberg,Pascale Richalet,Marc Vanhove,Jonas Stenberg

doi:10.1007/s00249-021-01560-2

Diana Spiegelberg, Pascale Richalet + Show 2 more

Open Access

https://doi.org/10.1007/s00249-021-01560-2

Copy DOI

Abstract

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.

Highlights

Cell surface receptors, including—but not limited to—Gprotein-coupled receptors (GPCRs), constitute the most successful class of target proteins for drug discovery research (Cacace et al 2003)
In addition to KD determination, special emphasis has been given to methodologies that give access to the binding kinetics, thereby allowing a description of the entire dynamic aspect of ligand–receptor interactions for the prospective design of drugs with improved safety and efficacy profiles, and with better defined binding mechanisms (Andersson et al 2006; Copeland et al 2006; Dahl and Akerud 2013; Georgi et al 2018; Schreiber 2002; Swinney 2008, 2009)
Despite a dissociation rate < 10–5 s−1, the dissociation phase showed a decrease of 14%, allowing robust koff estimation

Summary

Introduction

Cell surface receptors, including—but not limited to—Gprotein-coupled receptors (GPCRs), constitute the most successful class of target proteins for drug discovery research (Cacace et al 2003). The equilibrium dissociation constant KD describes the interaction of a drug or ligand L with a receptor R (with KD = [L].[R]/[LR]) and is a critical metric to characterize the binding affinity of ligand–receptor interactions. In addition to KD determination, special emphasis has been given to methodologies that give access to the binding kinetics, thereby allowing a description of the entire dynamic aspect of ligand–receptor interactions for the prospective design of drugs with improved safety and efficacy profiles, and with better defined binding mechanisms (Andersson et al 2006; Copeland et al 2006; Dahl and Akerud 2013; Georgi et al 2018; Schreiber 2002; Swinney 2008, 2009).

Methods

Results

Conclusion