Structural insights into epitope-paratope interactions of monoclonal antibody targeting CEACAM5-expressing tumors

Abstract

Abstract Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are overexpressed in some tumor types. The antibody-drug conjugate, tusamitamab ravtansine, specifically recognizes the A3-B3 domains of human CEACAM5 (hCEACAM5). To understand this specificity, we mapped the epitope-paratope interface between the A3-B3 domains of hCEACAM5 (hCEACAM5A3 − B3) and the antigen-binding fragment of tusamitamab (tusa Fab). Hydrogen/deuterium exchange mass spectrometry identified the tusa Fab paratope, which involves heavy chain (HC) residues 101–109 and light chain residues 48–54 and 88–104. Surface plasmon resonance demonstrated that alanine variants of HC residues 96–108 abolish binding to hCEACAM5, suggesting that these residues are critical for the Fab-antigen complex formation. Cryogenic electron microscopy structure of the tusa Fab-hCEACAM5A3 − B3 complex (3.11 Å overall resolution) revealed a discontinuous epitope involving residues in the A3-B3 domains and an N-linked mannose at residue 612. Conformational constraints on the epitope-paratope interface enable tusamitamab to target hCEACAM5A3 − B3 and differentiate it from other CEACAMs.