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.