title: Characterization and application of two RANK-specific antibodies with different biological activities.
abstract: Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools.
authors.affiliation: CNRS UPR 3572, Laboratory of Immunopathology and Therapeutic Chemistry, University of Strasbourg, Strasbourg 67000, France; Prestwick Chemical, Blvd Gonthier d'Andernach, Parc d'innovation, 67400 Illkirch, France.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Chypre
authors.affiliation: MedImmune, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Seaman
authors.affiliation: CNRS UPR 3572, Laboratory of Immunopathology and Therapeutic Chemistry, University of Strasbourg, Strasbourg 67000, France.
authors.forename: Olga G
authors.initials: OG
authors.lastname: Cordeiro
authors.affiliation: Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.
authors.forename: Laure
authors.initials: L
authors.lastname: Willen
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Knoop
authors.affiliation: MedImmune, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Buchanan
authors.affiliation: MedImmune, Granta Park, Cambridge CB21 6GH, UK.
authors.forename: Richard C A
authors.initials: RC
authors.lastname: Sainson
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Ifor R
authors.initials: IR
authors.lastname: Williams
authors.affiliation: Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Yagita
authors.affiliation: Department of Biochemistry, University of Lausanne, Epalinges, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Schneider
authors.affiliation: CNRS UPR 3572, Laboratory of Immunopathology and Therapeutic Chemistry, University of Strasbourg, Strasbourg 67000, France. Electronic address: c.mueller@ibmc-cnrs.unistra.fr.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Mueller
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000915:Antibody Affinity; D002454:Cell Differentiation / Q000187:drug effects; D061505:Cell Surface Display Techniques; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology*; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007117:Immunization, Secondary; D056747:Immunomodulation; D007422:Intestines / Q000166:cytology / Q000187:drug effects*; D019169:Jurkat Cells; D007801:Langerhans Cells / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D016328:NF-kappa B; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505687:Tnfrsf11a protein, mouse
keywords: Epithelial microfold cell; Langerhans cell; Monoclonal antibody; RANK (TNFRSF11a)