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The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.nanobody | CXCR4 antagonist | chemotaxis | HIV | stem cell mobilization

Neutralizing the interaction of the platelet receptor gpIb with VWF is an attractive strategy to treat and prevent thrombotic complications. ALX-0081 is a bivalent Nanobody which specifically targets the gpIb-binding site of VWF and interacts avidly with VWF. Nanobodies are therapeutic proteins derived from naturally occurring heavy-chain-only Abs and combine a small molecular size with a high inherent stability. ALX-0081 exerts potent IntroductionThe successive adhesion, activation, and aggregation of platelets are key processes in arterial thrombus formation after endothelial damage. 1,2 Both rupture of atherosclerotic plaques as well as surgical interventions to treat atherosclerosis (eg, percutaneous coronary intervention [PCI]) may cause exposure of the subendothelium and subsequent clot formation. Eventually, this can result in the occlusion of arteries, leading to ischemia, myocardial infarcts, or stroke. Given the central role of platelets in thrombosis, a substantial number of currently marketed antithrombotic drugs, such as aspirin, clopidogrel, and abciximab, target different steps involved in platelet activation and aggregation. 1,2 Thanks to their complementary mechanisms of action, the combination of these agents inhibits platelet aggregation to a greater extent than either agent alone. 3 However, the use of these antiplatelet drugs is hampered by an increased bleeding risk 1,2 and the occurrence of treatment resistance in some patients. 4 Moreover, the irreversible nature of their action can complicate the staunching of bleeding. 1,2 Inhibition of the initial adhesion of platelets to subendothelial collagen provides an alternative strategy to prevent unwanted clot formation. The plasma glycoprotein VWF plays a pivotal role in this adhesion via binding to exposed collagen on the one hand, and the interaction of its A1 domain with the gpIb-IX-V receptor complex on the surface of platelets on the other hand. [5][6][7] Interestingly, the VWF A1 domain is only exposed under high-shear conditions, 8,9 so VWF only acts as a bridging molecule between collagen and platelets in small or stenosed arteries. Therefore, it is expected that drugs inhibiting this interaction between VWF and platelets show an improved safety profile with respect to bleeding tendency. Indeed, the antithrombotic effect of several compounds targeting the gpIb-VWF-A1-axis, like aurintricarboxylic acid, 10-12 recombinant VWF fragments, 10,13-16 a recombinant gpIb chimeric protein, 17,18 anti-VWF mAbs, [19][20][21][22][23][24][25][26][27] and an anti-VWF aptamer 28 has been demonstrated in vitro and in vivo, without increasing the bleeding risk. 13,[16][17][18]21,23,25,28,29 Nevertheless, until now only 3 drug candidates have been evaluated in humans, including ALX-0081. [30][31][32][33] We developed ALX-0081, a bivalent humanized Nanobody directed against the A1 domain of VWF. Nanobodies are therapeutic proteins derived from the heavy-chain variable domains (VHH) that occur naturally in heavy-chain-only Igs of Camelidae. 34,35 Here we...

Soluble von Willebrand factor (VWF) has a low affinity for platelet glycoprotein (GP) Ib␣ and needs immobilization and/or high shear stress to enable binding of its A1 domain to the receptor. The previously described anti-VWF monoclonal antibody 1C1E7 enhances VWF/GPIb␣ binding and recognizes an epitope in the amino acids 764 -1035 region in the N-terminal DD3 domains. In this study we demonstrated that the DD3 region negatively modulates the VWF/GPIb-IX-V interaction; (i) deletion of the DD3 region in VWF augmented binding to GPIb␣, suggesting an inhibitory role for this region, (ii) the isolated DD3 region inhibited the GPIb␣ interaction of a VWF deletion mutant lacking this region, indicating that intramolecular interactions limit the accessibility of the A1 domain, (iii) using a panel of anti-VWF monoclonal antibodies, we next showed that the DD3 region is in close proximity with the A1 domain in soluble VWF but not when VWF was immobilized; (iv) destroying the epitope of 1C1E7 resulted in a mutant VWF with an increased affinity for GPIb␣. Our results support a model of domain translocation in VWF that allows interaction with GPIb␣. The suggested shielding interaction of the A1 domain by the DD3 region then becomes disrupted by VWF immobilization. The plasma protein von Willebrand factor (VWF)4 has a central role in normal primary hemostasis (1). The interaction of VWF with its platelet receptor glycoprotein (GP) Ib␣ in the GPIb-IX-V complex mediates platelet adhesion to extracellular matrices exposed at sites of vascular injury. This interaction is essential for thrombus formation at sites of high shear stress, as in microarterioles or in stenosed arteries.Mature VWF comprises a series of multimers that are composed of homodimers interlinked through disulfide bridges. The mature VWF subunit consists of four distinct types of internal homology present in two to three copies in the following order from the N terminus: