Integrated Drug Discovery, Sanofi R&D, Paris, France.
Large Molecules Research, Sanofi R&D, Paris, France.
Nat Commun. 2024 Oct 30;15(1):9377. doi: 10.1038/s41467-024-53746-9.
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 (hCEACAM5) 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 hCEACAM5- 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 hCEACAM5 and distinguish CEACAM5 from other CEACAMs.
癌胚抗原相关细胞粘附分子（CEACAMs）在一些肿瘤类型中过表达。抗体药物偶联物 tusamitamab ravtenzine 特异性识别人 CEACAM5（hCEACAM5）的 A3-B3 结构域。为了了解这种特异性，我们在这里绘制了 hCEACAM5（hCEACAM5）的 A3-B3 结构域与 tusamitamab（tusa Fab）的抗原结合片段之间的表位-抗体结合位界面。我们使用氢/氘交换质谱鉴定了 tusa Fab 的抗体结合位，该结合位涉及重链（HC）残基 101-109 和轻链残基 48-54 和 88-104。使用表面等离子体共振，我们证明 HC 残基 96-108 的丙氨酸变体丧失了与 hCEACAM5 的结合能力，表明这些残基对于 tusa-Fab-抗原复合物的形成至关重要。hCEACAM5-tusa Fab 复合物的低温电子显微镜结构（总体分辨率为 3.11Å）揭示了一个不连续的表位，涉及 A3-B3 结构域中的残基和残基 Asn612 上的 N-连接甘露糖。表位-抗体结合位界面的构象限制使 tusamitamab 能够靶向 hCEACAM5，并将 CEACAM5 与其他 CEACAMs 区分开来。
