title: Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action.
abstract: 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 chemokine 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 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 efficacy.
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: M E
authors.initials: ME
authors.lastname: Bradley
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.) steven.charlton@nottingham.ac.uk bruno.dombrecht@ablynx.com.
authors.forename: B
authors.initials: B
authors.lastname: Dombrecht
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: J
authors.initials: J
authors.lastname: Manini
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: J
authors.initials: J
authors.lastname: Willis
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: D
authors.initials: D
authors.lastname: Vlerick
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: S
authors.initials: S
authors.lastname: De Taeye
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: K
authors.initials: K
authors.lastname: Van den Heede
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: A
authors.initials: A
authors.lastname: Roobrouck
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: E
authors.initials: E
authors.lastname: Grot
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: T C
authors.initials: TC
authors.lastname: Kent
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: T
authors.initials: T
authors.lastname: Laeremans
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: S
authors.initials: S
authors.lastname: Steffensen
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: G
authors.initials: G
authors.lastname: Van Heeke
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: Z
authors.initials: Z
authors.lastname: Brown
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.) steven.charlton@nottingham.ac.uk bruno.dombrecht@ablynx.com.
authors.forename: S J
authors.initials: SJ
authors.lastname: Charlton
authors.affiliation: Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom (M.E.B., J.M., J.W., E.G., T.C.K., G.V.H., Z.B., S.J.C.); Ablynx NV, Zwijnaarde, Belgium (B.D., D.V., S.D.T., K.V.H., A.R., S.S., K.D.C.); and Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium (T.L.).
authors.forename: K D
authors.initials: KD
authors.lastname: Cromie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D016466:CHO Cells; D002161:Camelids, New World; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008969:Molecular Sequence Data; D023063:Receptors, Interleukin-8B / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D061905:Single-Domain Antibodies / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D023063:Receptors, Interleukin-8B; D061905:Single-Domain Antibodies