CryoEM structure of a therapeutic antibody (favezelimab) bound to human LAG3 determined using a bivalent Fab as fiducial marker

• Published in: Structure (London) 2023-10, Vol.31 (10), p.1149-1157.e3
• Main Authors: Mishra, Arjun K., Shahid, Salman, Karade, Sharanbasappa S., Agnihotri, Pragati, Kolesnikov, Alexander, Hasan, S. Saif, Mariuzza, Roy A.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - metabolism; antibody; Binding Sites; Cryoelectron Microscopy - methods; cryoEM; fiducial marker; Fiducial Markers; Humans; inhibitory receptor; LAG3; T cell; T-Lymphocytes - metabolism; X-ray crystallography
• ISSN: 0969-2126; 1878-4186; 1878-4186
• DOI: 10.1016/j.str.2023.07.013

Lymphocyte activation gene 3 protein (LAG3) is an inhibitory receptor that is upregulated on exhausted T cells in tumors. LAG3 is a major target for cancer immunotherapy with many anti-LAG3 antibodies in clinical trials. However, there is no structural information on the epitopes recognized by these antibodies. We determined the single-particle cryoEM structure of a therapeutic antibody (favezelimab) bound to LAG3 to 3.5 Å resolution, revealing that favezelimab targets the LAG3-binding site for MHC class II, its canonical ligand. The small size of the complex between the conventional (monovalent) Fab of favezelimab and LAG3 (∼100 kDa) presented a challenge for cryoEM. Accordingly, we engineered a bivalent version of Fab favezelimab that doubled the size of the Fab–LAG3 complex and conferred a highly identifiable shape to the complex that facilitated particle selection and orientation for image processing. This study establishes bivalent Fabs as new fiducial markers for cryoEM analysis of small proteins. [Display omitted] •LAG3 is a major target for cancer immunotherapy using anti-LAG3 antibodies•We determined the cryoEM structure of LAG3 bound to a therapeutic antibody•Structure determination required engineering a bivalent Fab to increase complex size•Bivalent Fabs are new fiducial markers for cryoEM analysis of small proteins LAG3 is receptor on T cells that is a major target for cancer immunotherapy using monoclonal antibodies against LAG3. However, it is unknown how these antibodies bind LAG3. Mishra et al. used cryoEM to identify the site on LAG3 targeted by a therapeutic antibody, which explains how the antibody works.