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, here we map the epitope-paratope interface between the A3-B3 domains of hCEACAM5 (hCEACAM5A3-B3) and the antigen-binding fragment of tusamitamab (tusa Fab). We use hydrogen/deuterium exchange mass spectrometry to identify the tusa Fab paratope, which involves heavy chain (HC) residues 101-109 and light chain residues 48-54 and 88-104. Using surface plasmon resonance, we demonstrate that alanine variants of HC residues 96-108 abolish binding to hCEACAM5, suggesting that these residues are critical for tusa-Fab-antigen complex formation. The cryogenic electron microscopy structure of the hCEACAM5A3-B3- tusa Fab complex (3.11 Å overall resolution) reveals a discontinuous epitope involving residues in the A3-B3 domains and an N-linked mannose at residue Asn612. Conformational constraints on the epitope-paratope interface enable tusamitamab to target hCEACAM5A3-B3 and distinguish CEACAM5 from other CEACAMs.