CXCR4-Specific Nanobodies as Potential Therapeutics for WHIM syndrome

Raymond H de Wit, Raimond Heukers, Hendrik J Brink, Angela Arsova, David Maussang, Pasquale Cutolo, Beatrijs Strubbe, Henry F Vischer, Françoise Bachelerie, Martine J Smit

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WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: warts, hypogammaglobulinemia, infections, and myelokathexis, which refers to abnormal accumulation of mature neutrophils in the bone marrow. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4, giving these CXCR4-WHIM mutants a gain of function in response to their ligand CXCL12. Considering the broad functions of CXCR4 in maintaining leukocyte homeostasis, patients are panleukopenic and display altered immune responses, likely as a consequence of impairment in the differentiation and trafficking of leukocytes. Treatment of WHIM patients currently consists of symptom relief, leading to unsatisfactory clinical responses. As an alternative and potentially more effective approach, we tested the potency and efficacy of CXCR4-specific nanobodies on inhibiting CXCR4-WHIM mutants. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies. They combine the advantages of small-molecule drugs and antibody-based therapeutics due to their relative small size, high stability, and high affinity. We compared the potential of monovalent and bivalent CXCR4-specific nanobodies to inhibit CXCL12-induced CXCR4-WHIM-mediated signaling with the small-molecule clinical candidate AMD3100. The CXCR4-targeting nanobodies displace CXCL12 binding and bind CXCR4-wild type and CXCR4-WHIM (R334X/S338X) mutants and with (sub-) nanomolar affinities. The nanobodies' epitope was mapped to extracellular loop 2 of CXCR4, overlapping with the binding site of CXCL12. Monovalent, and in particular bivalent, nanobodies were more potent than AMD3100 in reducing CXCL12-mediated G protein activation. In addition, CXCR4-WHIM-dependent calcium flux and wound healing of human papillomavirus-immortalized cell lines in response to CXCL12 was effectively inhibited by the nanobodies. Based on these in vitro results, we conclude that CXCR4 nanobodies hold significant potential as alternative therapeutics for CXCR4-associated diseases such as WHIM syndrome.

Original languageEnglish
Pages (from-to)35-44
Number of pages10
JournalThe Journal of Pharmacology and Experimental Therapeutics
Issue number1
Publication statusPublished - 1 Oct 2017


The authors thank David McDermott and Philip Murphy for the discussions and providing the K562 cell lines. H.F.V., F.B., and M.J.S. are part of the European Union’s Horizon 2020 MSCA Programme under Grant Agreement 641833 (ONCORNET). This work was supported by the Netherlands Organization for Scientific Research [Vici Grant] and the Dutch Technology Foundation. 1Current affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. 2Current affiliation: Merus N.V., Utrecht, The Netherlands. s This article has supplemental material available at

FundersFunder number
European Union’s Horizon 2020 MSCA
Horizon 2020 Framework Programme641833
Nederlandse Organisatie voor Wetenschappelijk Onderzoek
Stichting voor de Technische Wetenschappen


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