Abstract
Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacological characterization of Nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent Nanobodies were identified and detailed epitope mapping showed that these bind to distinct, non-overlapping epitopes on the CXCR2 receptor. The N-terminal or class 1 monovalent Nanobodies possessed higher potencies but lacked efficacy at high agonist concentrations. In contrast, the extracellular loop or class 2 monovalent Nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking GRO-α and IL-8 mediated CXCR2 signalling, both classes of Nanobodies also displayed inverse agonist behaviour. Bivalent and biparatopic Nanobodies were generated, respectively combining Nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic Nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic Nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and/or efficacy.
- Chemotactic peptides
- Receptor dimerization
- Interleukins
- Gi family
- Func. analysis receptor/ion channel mutants
- Fluorescence techniques
- Receptor binding studies
- Leukocytes/Mast cells
- The American Society for Pharmacology and Experimental Therapeutics