title: Increment in Drug Loading on an Antibody-Drug Conjugate Increases Its Binding to the Human Neonatal Fc Receptor in Vitro.
abstract: Antibody-drug conjugates, such as brentuximab vedotin (BTXv), are an innovative category of monoclonal antibodies. BTXv is bioconjugated via the chemical reduction of cysteine residues involved in disulfide bonds. Species of BTXv containing zero, two, four, six, or eight vedotin molecules per antibody coexist in the stock solution. We investigated the influence of drug loading on the binding of the antibody to FcRn, a major determinant of antibody pharmacokinetics in humans. We developed a hydrophobic interaction chromatography (HIC) method for separating the different species present in the stock solution of BTXv, and we purified and characterized the collected species before use. We assessed the binding of these different species to FcRn in a cellular assay based on flow cytometry and surface plasmon resonance. HIC separated the different species of BTXv and allowed their collection at adequate levels of purity. Physicochemical characterization showed that species with higher levels of drug loading tended to form more aggregates. FcRn binding assays showed that the most conjugated species, particularly those with saturated loading, interacted more strongly than unconjugated BTXv with the FcRn.
authors.affiliation: CNRS, GICC UMR 7292, CHRU de Tours, Service d'Immunologie, Université François Rabelais de Tours , Tours, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Brachet
authors.affiliation: CNRS, GICC UMR 7292, CHRU de Tours, Service de Pharmacie, Université François-Rabelais de Tours , Tours, France.
authors.forename: Renaud
authors.initials: R
authors.lastname: Respaud
authors.affiliation: CNRS, GICC UMR 7292, Université François Rabelais de Tours , Tours, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Arnoult
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM, U1194, Université Montpellier, ICM Institut Régional du Cancer , Montpellier, F-34090 France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Henriquet
authors.affiliation: CNRS, GICC UMR 7292, Université François Rabelais de Tours , Tours, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Dhommée
authors.affiliation: CNRS, GICC UMR 7292, Université François Rabelais de Tours , Tours, France.
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Viaud-Massuard
authors.affiliation: INSERM U1100, Université François Rabelais de Tours , Tours, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Heuze-Vourc'h
authors.affiliation: CNRS, GICC UMR 7292, Université François Rabelais de Tours , Tours, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Joubert
authors.affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM, U1194, Université Montpellier, ICM Institut Régional du Cancer , Montpellier, F-34090 France.
authors.forename: Martine
authors.initials: M
authors.lastname: Pugnière
authors.affiliation: CNRS, GICC UMR 7292, CHRU de Tours, Service d'Immunologie, Université François Rabelais de Tours , Tours, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Gouilleux-Gruart
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000079963:Brentuximab Vedotin; D002850:Chromatography, Gel; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018796:Immunoconjugates / Q000378:metabolism; D011485:Protein Binding; D011961:Receptors, Fc / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D018796:Immunoconjugates; D011961:Receptors, Fc; D000079963:Brentuximab Vedotin; C100688:Fc receptor, neonatal
keywords: FcRn; antibody−drug conjugate; drug-to-antibody ratio; flow cytometry; hydrophobic interaction chromatography; surface plasmon resonance