Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists.

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

Antibodies Targeting Chemokine Receptors CXCR4 and ACKR3.

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 interest from academic groups and pharmaceutical companies, there are currently few approved medicines targeting chemokine receptors. Monoclonal antibodies (mAbs) 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 CXCR4 and 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 utilization 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 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.

Differential effects of FcRn antagonists on the subcellular trafficking of FcRn and albumin.

The homeostasis of IgG is maintained by the neonatal Fc receptor, FcRn. Consequently, antagonism of FcRn to reduce endogenous IgG levels is an emerging strategy for treating antibody-mediated autoimmune disorders using either FcRn-specific antibodies or an engineered Fc fragment. For certain FcRn-specific antibodies, this approach has resulted in reductions in the levels of serum albumin, the other major ligand transported by FcRn. Cellular and molecular analyses of a panel of FcRn antagonists have been carried out to elucidate the mechanisms leading to their differential effects on albumin homeostasis. These analyses have identified 2 processes underlying decreases in albumin levels during FcRn blockade: increased degradation of FcRn and competition between antagonist and albumin for FcRn binding. These findings have potential implications for the design of drugs to modulate FcRn function.

Multivalent CXCR4-targeting nanobody formats differently affect affinity, receptor clustering, and antagonism.

The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or ß-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.

Correction: Zakrzewicz et al. Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus? Cells 2022, 11, 942.

The authors wish to make the following changes to their paper [...].

Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

Pemphigus vulgaris is an autoimmune blistering disease of the epidermis, caused by autoantibodies against desmosomal proteins, mainly desmogleins 1 and 3, which induce an impairment of desmosomal adhesion and blister formation. Recent findings have shown that inhibition of immunoglobulin G binding on the neonatal Fc receptor, FcRn, results in reduced autoantibody recycling and shortens their half-life, providing a valid treatment option for PV. We have here analyzed the role of FcRn in human keratinocytes treated with antibodies isolated from pemphigus vulgaris patient or with recombinant anti-desmoglein-3 antibodies that induce pathogenic changes in desmosomes, such as loss of monolayer integrity, aberrant desmoglein-3 localization and degradation of desmoglein-3. We show that blocking IgG binding on FcRn by efgartigimod, a recombinant Fc fragment undergoing clinical studies for pemphigus, stabilizes the keratinocyte monolayer, whereas the loss of desmoglein-3 is not prevented by efgartigimod. Our data show that FcRn may play a direct role in the pathogenesis of pemphigus at the level of the autoantibody target cells, the epidermal keratinocytes. Our data suggest that in keratinocytes, FcRn may have functions different from its known function in IgG recycling. Therefore, stabilization of keratinocyte adhesion by FcRn blocking entities may provide a novel treatment paradigm for pemphigus.

Conformational selection guides ß-arrestin recruitment at a biased G protein-coupled receptor.

G protein-coupled receptors (GPCRs) recruit ß-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 ß-arrestins but not G proteins, making them a model system for investigating the structural basis of ß-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on 13CH3-ε-methionine-labeled ACKR3, revealing that ß-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular ß-arrestin-coupling region. NMR studies of ACKR3 mutants defective in ß-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 ß-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Nanobodies: New avenues for imaging, stabilizing and modulating GPCRs.

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.

Natural Killer Cell Hypo-responsiveness in Chronic Lymphocytic Leukemia can be Circumvented In Vitro by Adequate Activating Signaling.

Chronic lymphocytic leukemia (CLL) is characterized by an acquired immune dysfunction, which may underlie the hampered efficacy of cellular immunotherapy. Most data on dampened immune responses in CLL come from studies investigating CLL and T cell interactions. Natural killer (NK) cells may be an attractive alternative source of effector cells in immunotherapy in CLL, provided that functionality is retained within the CLL micro-environment. Despite their important role in anti-tumor responses, NK cells are not extensively characterized in CLL. Here, we studied the expression of activating and inhibitory receptors on CLL-derived and healthy control (HC) NK cells, and their functional response towards several stimuli. NK cells from CLL patients have an increased maturation stage, with an expansion of NKG2C+ NK cells in CMV seropositive individuals. The cytotoxicity receptor NKG2D is downregulated, and the killing capacity through this receptor was markedly reduced in CLL-derived NK cells. In contrast, activation via CD16 (FCγRIII) led to adequate activation and functional responses in CLL-derived NK cells. These findings indicate that NK cells in CLL are not intrinsically defect and still perform effector functions upon adequate activating signaling. Clinical relevance of this finding was shown by treatment with novel nanobody-Fc constructs, which induced cytotoxic responses in both CLL- and HC-derived NK cells via CD16. Our results show that NK cells, in contrast to the T cell compartment, retain their function within the CLL micro-environment, provided that they receive an adequate activating signal. These findings warrant future studies on NK cell mediated immunotherapeutic strategies in CLL.

Display Technologies for Generation of Ig Single Variable Domains.

Variable fragments of heavy-chain-only antibodies (VHH) found in camelids are valuable research tools in pharmacology and biotechnology and are being developed for the clinic to treat patients with autoimmune and infectious diseases or cancer. Their single-domain nature and biochemical properties greatly facilitate the development process. The most common technology to select single-domain antibody fragments is phage display following active immunization of llamas or other members of Camelidae family. Selection of VHH from immune phage libraries is a rapid approach to discover a broad panel of in vivo matured antigen-specific clones with comprehensive functionalities. In this chapter, we describe a detailed protocol for construction of VHH immune libraries and phage display selection against antigens in their native conformation.

Nanobody-Fc constructs targeting chemokine receptor CXCR4 potently inhibit signaling and CXCR4-mediated HIV-entry and induce antibody effector functions.

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.

CXCR4-targeting nanobodies differentially inhibit CXCR4 function and HIV entry.

The chemokine receptor CXCR4 and its ligand CXCL12 contribute to a variety of human diseases, such as cancer. CXCR4 is also a major co-receptor facilitating HIV entry. Accordingly, CXCR4 is considered as an attractive therapeutic target. Drug side effects and poor pharmacokinetic properties have been major hurdles that have prevented the implementation of CXCR4-directed inhibitors in treatment regimes. We evaluated the activity of a new and promising class of biologics, namely CXCR4-targeting nanobodies, with the purpose of identifying nanobodies that would preferentially inhibit HIV infection, while minimally disturbing other CXCR4-related functions. All CXCR4-interacting nanobodies inhibited CXCL12 binding and receptor-mediated calcium mobilization with comparable relative potencies. Importantly, the anti-HIV-1 activity of the nanobodies did not always correlate with their ability to modulate CXCR4 signaling and function, indicating that the anti-HIV and anti-CXCR4 activity are not entirely overlapping and may be functionally separated. Three nanobodies with divergent activity profiles (VUN400, VUN401 and VUN402) were selected for in depth biological evaluation. While all three nanobodies demonstrated inhibitory activity against a wide range of HIV (X4) strains, VUN402 poorly blocked CXCL12-induced CXCR4 internalization, chemotaxis and changes in cell morphology. Each of these nanobodies recognized distinct, although partially overlapping epitopes on CXCR4, which might underlie their distinct activity profiles. Our results demonstrate the potential of CXCR4-targeting nanobody VUN402 as a novel lead and starting point for the development of a more potent and selective anti-HIV agent.

Recombinant C1 Esterase Inhibitor Reduces Cytokine Storm in an Ex Vivo Whole Blood Model.

C1 esterase inhibitor (C1INH) is an abundant component of blood plasma (the average concentration is 250 mg/L); it is known to be involved in several biological processes, for instance, in the regulation of the coagulation system, adhesion of leukocytes on endothelial cells, and in the regulation of complement and kallikrein cascades. Lately, the role of C1INH in immunomodulation has gained considerable attention. We used an ex vivo whole blood model to examine the influence of C1INH and its mutated variants on the inflammatory cytokines interleukin (IL)-6, tumor necrosis factor-α (TNFα), and IL-1ß. The present study demonstrated for the first time that recombinant C1INH or its Seprin domain can downregulate bacterial endotoxin induced IL-6 release. We also observed that unstructured N-terminal domain of C1INH downregulates the release of IL-1ß and TNFα, but not IL-6. Our results suggest that C1INH may have therapeutic potential for treatment of inflammatory conditions.

DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.

The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.