Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen–Deuterium Exchange Mass Spectrometry

Hydrogen–deuterium
exchange mass spectrometry (HDX-MS) is
a method to probe the solvent accessibility and conformational dynamics
of a protein or a protein–ligand complex with respect to exchangeable
amide hydrogens. Here, we present the application of HDX-MS to determine
the binding sites of Affimer reagents to the monoclonal antibodies
trastuzumab and pertuzumab, respectively. Intact and subunit level
HDX-MS analysis of antibody-affimer complexes showed significant protection
from HDX in the antibody Fab region upon affimer binding. Bottom-up
HDX-MS experiments including online pepsin digestion revealed that
the binding sites of the affimer reagents were mainly located in the
complementarity-determining region (CDR) 2 of the heavy chain of the
respective antibodies. Three-dimensional models of the binding interaction
between the affimer reagents and the antibodies were built by homology
modeling and molecular docking based on the HDX data.