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

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.

Site‐Selective Protein Conjugation by a Multicomponent Ugi Reaction

“…Starting from our E1‐selective Ugi‐conjugated trastuzumab, we thus engaged it in a SPAAC step with a BCN‐fluorescein partner, effectively delivering the fluorescent conjugate T Ugi ‐Fluo (Figure 3). Gratifyingly, the latter showed rate constants of association and dissociation identical to those of the control FITC‐labeled trastuzumab T‐Fluo on HER2‐positive SKBR‐3 cells (i. e., k on =2.3×10 4 M −1 .s −1 , k off =1×10 −5 s −1 , K D =0.45 nM versus k on =2.0×10 4 M −1 .s −1 , k off =1.37×10 −5 s −1 , K D =0.69 nM, respectively), perfectly in line with previously reported values [38] . As further evidence of a maintained affinity, the ADC T Ugi ‐DM1 was produced by SPAAC, using a BCN‐DM1 strained alkyne (avDAR=1.0, 60–65 % conversion, 75 % yield due to partial precipitation of the ADC).…”

“…Gratifyingly, the latter showed rate constants of association and dissociation identical to those of the control FITClabeled trastuzumab T-Fluo on HER2-positive SKBR-3 cells (i. e., k on = 2.3×10 4 M À 1 .s À 1 , k off = 1×10 À 5 s À 1 , K D = 0.45 nM versus k on = 2.0×10 4 M À 1 .s À 1 , k off = 1.37×10 À 5 s À 1 , K D = 0.69 nM, respectively), perfectly in line with previously reported values. [38] As further evidence of a maintained affinity, the ADC T Ugi -DM1 was produced by SPAAC, using a BCN-DM1 strained alkyne (avDAR = 1.0, 60-65 % conversion, 75 % yield due to partial precipitation of the ADC). This ADC was tested in cell viability assays on both HER2-positive and HER2-negative cell lines (i. e., SKBR-3 and MDA-MB-231, respectively) against the marketed ADC Kadcyla, comprising the same linker-drug payload, but produced through stochastic lysine conjugation (avDAR = 3.5).…”

Binding Affinity of Trastuzumab and Pertuzumab Monoclonal Antibodies to Extracellular HER2 Domain

“…They observed a slightly preferential binding for trastuzumab over pertuzumab in both antibody forms, namely, Fab or the complete IgG form [5]. Furthermore, Spiegelberg et al determined dissociation constants for HER2/trastuzumab and HER2/pertuzumab complexes using the ligand tracer technique on living cell lines [6]. Their study revealed ∆G bind values of −13.7 and −14.0 kcal•mol −1 for the trastuzumab and pertuzumab complexes, respectively, at 310 K.…”