Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action

Bradley, M. E.; Dombrecht, B.; Manini, J.; Willis, J.; Vlerick, D.; De Taeye, S.; Van den Heede, K.; Roobrouck, A.; Grot, E.; Kent, T. C.; Laeremans, T.; Steffensen, S.; Van Heeke, G.; Brown, Z.; Charlton, S. J.; Cromie, K. D.

Abstract

Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics. Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemo-kine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding 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 CXCR2 signaling mediated by CXCL1 (growth-related oncogene α) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. 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 nano-bodies 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 efficacy.

Citation

Bradley, M. E., Dombrecht, B., Manini, J., Willis, J., Vlerick, D., De Taeye, S., …Cromie, K. D. (2015). Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action. Molecular Pharmacology, 87(2), 251-262. https://doi.org/10.1124/mol.114.094821

• Journal Article Type: Article
• Acceptance Date: Dec 2, 2014
• Online Publication Date: Dec 2, 2014
• Publication Date: 2015-02
• Deposit Date: Mar 16, 2018
• Journal: Molecular Pharmacology
• Print ISSN: 0026-895X
• Electronic ISSN: 1521-0111
• Publisher: American Society for Pharmacology and Experimental Therapeutics
• Peer Reviewed: Peer Reviewed
• Volume: 87
• Issue: 2
• Pages: 251-262
• DOI: https://doi.org/10.1124/mol.114.094821
• Public URL: https://nottingham-repository.worktribe.com/output/1107685
• PMID: 00034781