Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches.
Fiche publication
Date publication
septembre 2017
Journal
mAbs
Auteurs
Membres identifiés du Cancéropôle Est :
Dr CIANFERANI Sarah
Tous les auteurs :
Terral G, Champion T, Debaene F, Colas O, Bourguet M, Wagner-Rousset E, Corvaia N, Beck A, Cianferani S
Lien Pubmed
Résumé
Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.
Mots clés
epitope mapping, hydrogen/deuterium exchange mass spectrometry, mAb/antigen complexes, monoclonal antibody, native mass spectrometry
Référence
MAbs. 2017 Sep;:0