Development by Genetic Immunization of Monovalent Antibodies (Nanobodies) Behaving as Antagonists of the Human ChemR23 Receptor.

TitleDevelopment by Genetic Immunization of Monovalent Antibodies (Nanobodies) Behaving as Antagonists of the Human ChemR23 Receptor.
Publication TypeJournal Article
Year of Publication2016
AuthorsPeyrassol, X., T. Laeremans, M. Gouwy, V. Lahura, M. Debulpaep, J. Van Damme, J. Steyaert, M. Parmentier, and I. Langer
JournalJ Immunol
Volume196
Issue6
Pagination2893-901
Date Published2016 Mar 15
ISSN1550-6606
KeywordsAnimals, Antibodies, Monoclonal, Calcium Signaling, Camelids, New World, Cell Surface Display Techniques, Cells, Cultured, Chemokines, Dendritic Cells, DNA, Genetic Engineering, Humans, Immunization, Intercellular Signaling Peptides and Proteins, Macrophages, Mice, Peptide Fragments, Protein Binding, Receptors, Chemokine, Single-Domain Antibodies
Abstract

The generation of Abs that recognize the native conformation of G protein-coupled receptors can be a challenging task because, like most multimembrane-spanning proteins, they are extremely difficult to purify as native protein. By combining genetic immunization, phage display, and biopanning, we identified two functional monovalent Abs (nanobodies) targeting ChemR23. The two nanobodies (CA4910 and CA5183) were highly specific for the human receptor and bind ChemR23 with moderate affinity. Binding studies also showed that they share a common binding site that overlaps with that of chemerin, the natural ligand of ChemR23. Consistent with these results, we found that the nanobodies were able to antagonize chemerin-induced intracellular calcium increase. The inhibition was partial when chemerin was used as agonist and complete when the chemerin(149-157) nonapeptide was used as agonist. Engineering of a bivalent CA4910 nanobody resulted in a relatively modest increase in affinity but a marked enhancement of efficacy as an antagonist of chemerin induced intracellular calcium mobilization and a much higher potency against the chemerin(149-157) nonapeptide-induced response. We also demonstrated that the fluorescently labeled nanobodies detect ChemR23 on the surface of human primary cell populations as efficiently as a reference mouse mAb and that the bivalent CA4910 nanobody behaves as an efficient antagonist of chemerin-induced chemotaxis of human primary cells. Thus, these nanobodies constitute new tools to study the role of the chemerin/ChemR23 system in physiological and pathological conditions.

DOI10.4049/jimmunol.1500888
Alternate JournalJ. Immunol.
PubMed ID26864035
Research group: