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

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.

Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein–Protein Interaction Analysis, and Protein Function Exploration

“…Using a tertiary fluorescently labeled antibody, chromatin-specific staining was observed in human HCT116 cells and even in organisms evolutionarily distant from mammals (9). Peyrassol and colleagues developed His-tagged ChemR23 G-protein-coupled receptor (GPCR) nanobodies and tested their binding specificity by immunostaining on fixed CHO cells (21). Visualization was performed by using a fluorescently labeled anti-His secondary antibody, hence avoiding the use of a tertiary antibody (21).…”

“…Peyrassol and colleagues developed His-tagged ChemR23 G-protein-coupled receptor (GPCR) nanobodies and tested their binding specificity by immunostaining on fixed CHO cells (21). Visualization was performed by using a fluorescently labeled anti-His secondary antibody, hence avoiding the use of a tertiary antibody (21). …”

“…The aforementioned ChemR23 nanobodies uniquely recognize ChemR23 GPCR and antagonize chemerin-induced receptor activation. As chemerin also binds other GPCRs, the nanobodies can be used to discriminate ChemR23-specific signaling from other chemerin-induced pathways (21). …”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Camelid V H Hs generated against the Kv1.3 ion channel targeted extracellular loops, not the channel cavity, 85 and the epitopes of V H Hs against the P2X7 ion channel were not defined. 86 Similarly, camelid V H Hs developed as potential therapeutics against the chemokine receptors CXCR4, 84 CXCR7 87 and ChemR23, 88 as well as V H Hs used as crystallization chaperones for several GPCRs, channels and transporters, 89–95 all appear to bind solvent-exposed extracellular or intracellular loops of these receptors in a manner similar to conventional antibodies and their fragments. By contrast, a synthetic CXCR4-binding “i-body” engineered from an Ig-like NCAM domain was found to penetrate deep into the receptor’s ligand-binding pocket to occupy a truly cryptic, partially transmembrane epitope.…”

Antibodies targeting G protein-coupled receptors: Recent advances and therapeutic challenges

“…The bivalent Nb was much more efficient as an antagonist, and inhibited chemerin-induced chemotaxis of human monocyte-derived dendritic cells. 24 Altogether, the use of these Nbs and previously generated mouse mAbs support the existence of 2 independent binding sites for chemerin, one for the cystatin-like domain, the other for the C-terminal peptide that triggers receptor activation. The new Nbs also constitute interesting tools to study the role of the chemerin/ CMKLR1 system in physiology and diseases.…”