Upregulation of the chemokine receptor CXCR4 contributes to the progression and metastasis of both solid and hematological malignancies, rendering this receptor an attractive therapeutic target. Besides the only FDA-approved CXCR4 antagonist Plerixafor (AMD3100), multiple other classes of CXCR4-targeting molecules are under (pre-)clinical development. Nanobodies (Nb), small single variable domains of heavy-chain only antibodies from Camelids, have appeared to be ideal antibody-fragments for targeting a broad range of epitopes and cavities within GPCRs such as CXCR4. Compared to conventional antibodies, monovalent nanobodies show fast blood clearance and no effector functions. In order to further increase their binding affinities and to restore antibody-mediated effector functions, we have constructed three different bivalent nanobody Fc-fusion molecules (Nb-Fc), targeting distinct epitopes on CXCR4, via fusion of Nbs to a Fc domain of a human IgG1 antibody. Most Nb-Fc constructs show increased binding affinity and enhanced potency in CXCL12 displacement, inhibition of CXCL12-induced signaling and CXCR4-mediated HIV entry, when compared to their monovalent Nb counterparts. Moreover, Nb-Fc induced ADCC- and CDC-mediated cell-death of CXCR4-overexpressing CCRF-CEM leukemia cells and did not affect cells expressing low levels or no CXCR4. These highly potent CXCR4 Nb-Fc constructs with Fc-mediated effector functions are attractive molecules to therapeutically target CXCR4-overexpressing tumors.

Significance: Class A G protein−coupled receptors (GPCRs) can form dimers and oligomers via poorly understood mechanisms. We show here that the chemokine receptor CXCR4, which is a major pharmacological target, has an oligomerization behavior modulated by its active conformation. Combining advanced, single-molecule, and single-cell optical tools with functional assays and computational approaches, we unveil three key features of CXCR4 quaternary organization: CXCR4 dimerization 1) is dynamic, 2) increases with receptor expression level, and 3) can be disrupted by stabilizing an inactive receptor conformation. Ligand binding motifs reveal a ligand binding subpocket essential to modulate both CXCR4 basal activity and dimerization. This is relevant to develop new strategies to design CXCR4-targeting drugs.

The family of G protein-coupled receptors (GPCRs) is the largest class of membrane proteins and an important drug target due to their role in many (patho)physiological processes. Besides small molecules, GPCRs can be targeted by biologicals including antibodies and antibody fragments. This review describes the use of antibodies and in particular antibody fragments from camelid-derived heavy chain-only antibodies (nanobodies/VHHs/sdAbs) for detecting, stabilizing, modulating and therapeutically targeting GPCRs. Altogether, it becomes increasingly clear that the small size, structure and protruding antigen-binding loops of nanobodies are favorable features for the development of selective and potent GPCRs-binding molecules. This makes them attractive tools to modulate GPCR activity but also as targeting modalities for GPCR-directed therapeutics. In addition, these antibody-fragments are important tools in the stabilization of particular conformations of these receptors. Lastly, nanobodies, in contrast to conventional antibodies, can also easily be expressed intracellularly which render nanobodies important tools for studying GPCR function. Hence, GPCR-targeting nanobodies are ideal modalities to image, stabilize and modulate GPCR function.

G protein–coupled receptors (GPCRs) recruit β-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage β-arrestins but not G proteins, making them a model system for investigating the structural basis of β-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on ¹³ CH ₃ -ε–methionine–labeled ACKR3, revealing that β-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular β-arrestin–coupling region. NMR studies of ACKR3 mutants defective in β-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides β-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Dysregulation of the chemokine system is implicated in a number of autoimmune and inflammatory diseases, as well as cancer. Modulation of chemokine receptor function is a very promising approach for therapeutic intervention. Despite an interest from academic groups and pharmaceutical companies, there are currently few approved medicines targeting chemokine receptors. Monoclonal antibodies (mAb) and antibody-based molecules have been successfully applied in the clinical therapy of cancer and represent a potential new class of therapeutics targeting chemokine receptors, belonging to the class of G protein-coupled receptors (GPCRs). Besides conventional mAbs, single-domain antibodies and antibody scaffolds are also gaining attention as promising therapeutics. In this review we provide an extensive overview of mAbs, single-domain antibodies and other antibody fragments targeting the C-X-C chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7. We discuss their unique properties and advantages over small molecule compounds, and also refer to the molecules in preclinical and clinical development. We focus on single-domain antibodies and scaffolds and their utilisation in GPCR research. Additionally, structural analysis of antibody binding to CXCR4 is discussed. SIGNIFICANCE STATEMENT Modulating the function of GPCRs and, particularly, chemokine receptors draws a high interest. A comprehensive review is provided for monoclonal antibodies, antibody fragments and variants directed at CXCR4 and ACKR3. Their advantageous functional properties, versatile applications as research tools and use in the clinic are discussed.