Integrated Approach for Characterizing Bispecific Antibody/Antigens Complexes and Mapping Binding Epitopes with SEC/MALS, Native Mass Spectrometry, and Protein Footprinting

Abstract

Bispecific antibodies (BsAbs), with a unique mechanism of recognizing two different epitopes or antigens, have shown potential in various therapeutic areas. Molecular characterization of BsAbs’ epitopes not only allows for detailed understanding of their mechanism of actions but also guides the design and selection of drug candidate molecules. In this study, we illustrate the practical utility of an integrated approach, including size exclusion chromatography with multiangle light scattering and native mass spectrometry (MS) for the biophysical characterization of complex formation of a BsAb with two target antigens, cluster of differentiation 3 (CD3) and B-cell maturation antigen (BCMA). MS-based protein footprinting strategies, including hydrogen/deuterium exchange MS, fast photochemical oxidation of proteins, and carboxyl group footprinting with glycine ethyl ester, were further applied to determine BsAb’s binding epitopes. This combination approach provides molecular details on the binding mechanisms of BsAb to the two distinct antigens with rapid output and high resolution.

Structure of the human marker of self 5-transmembrane receptor CD47

“…Using that model as a reference, we aligned the Cα atoms of the polypeptide chain with the Cα atoms of the ECD (residues 1-115) from the ECD-B6H12 complex structure with PDB ID 5TZU [https://doi.org/ 10.2210/pdb5TZU/pdb] using COOT 37 Hydrogen/deuterium exchange mass spectrometry. HDX-MS experiments were conducted as described previously 42 . In brief, HDX was conducted on an HDX PAL robot (LEAP Technologies, Carrboro, NC) where 5 μL of each protein construct at 1 mg/mL was diluted into 55 μL of deuterium oxide (D 2 O) buffer (10 mM phosphate buffer, 0.05% DDM, D 2 O, pD 7.0) to start the labeling reactions.…”

“…HDX-MS experiments were conducted as described previously 42 . In brief, HDX was conducted on an HDX PAL robot (LEAP Technologies, Carrboro, NC) where 5 μL of each protein construct at 1 mg/mL was diluted into 55 μL of deuterium oxide (D 2 O) buffer (10 mM phosphate buffer, 0.05% DDM, D 2 O, pD 7.0) to start the labeling reactions.…”

Humanization of a strategic CD3 epitope enables evaluation of clinical T-cell engagers in a fully immunocompetent in vivo model

“…HDX-MS was conducted as described previously 24 . In brief, non-deuterated experiments were performed to generate a list of common peptides for CD3 prior to epitope mapping experiments.…”

Label-free methods for opticalin vitrocharacterization of protein–protein interactions

“…Although used in the past to quantify binding affinities of proteins 6,93 publications from 2017 onwards suggest that SEC-MALS is now mostly used to assess purity, molecular weight, stoichiometry 105 and aggregation. In these publications, SEC-MALS is then complemented by techniques such as SPR, [106][107][108] MST, 106 ITC, [109][110][111] AUC, 92 fluorescence spectroscopy 112 and flow cytometry 113 to quantify binding affinities.…”