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Chemokine stimulation of atypical chemokine receptor 3 (ACKR3) does not activate G proteins but recruits arrestins. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that high constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive action of ACKR3 using novel inverse agonistic nanobodies to suppress basal activity. These new tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal, non-chemotactic, breast cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide a new therapeutic approach for targeting ACKR3., Competing Interests: Competing interests B.F.V. has an ownership interest in Protein Foundry, L.L.C. and XLock Biosciences, Inc. R.H. is affiliated with QVQ Holding BV. All other authors declare no competing interests.

HIV-1 alters the dynamics and distribution of tetraspanins, a group of proteins integral to membrane organization, to facilitate both entry and egress. Notably, the tetraspanin CD9 is dysregulated during HIV-1 infection, correlating with multifaceted effects on viral replication. Here, we generated llama-derived nanobodies against CD9 to restrict HIV-1 replication. We immunized llamas with recombinant large extracellular loop of CD9 and identified eight clonally distinct nanobodies targeting CD9, each exhibiting a range of affinities and differential binding to cell surface-expressed CD9. Notably, nanobodies T2C001 and T2C002 demonstrated low nanomolar affinities and exhibited differential sensitivities against endogenous and overexpressed CD9 on the cell surface. Although CD9-directed nanobodies did not impede the early stages of HIV-1 life cycle, they effectively inhibited virus-induced syncytia formation and virus replication in T cells and monocyte-derived macrophages. This discovery opens new avenues for host-targeted therapeutic strategies, potentially augmenting existing antiretroviral treatments for HIV-1., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

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 Gα i - or β-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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stephanie M. Anbuhl reports financial support was provided by European Commission Marie Sklodowska-Curie Actions. Angela I. Schriek reports financial support was provided by Aids Fund. Steven W. de Taeye reports financial support was provided by Health Holland. Tom N. Grossmann reports was provided by European Research Council. Saskia Neubacher reports financial support was provided by Horizon 2020 European Innovation Council Transition Open Program. Raimond Heukers reports a relationship with QVQ holding BV that includes: board membership, employment, and equity or stocks. Stephanie M. Anbuhl reports a relationship with QVQ Holding BV that includes: employment. Tom N. Grossmann reports a relationship with Incircular B.V. that includes: board membership, consulting or advisory, and equity or stocks. Saskia Neubacher reports a relationship with Incircular B.V. that includes: board membership, consulting or advisory, and equity or stocks. Tom N. Grossmann has patent #WO2019203645A1 pending to Stichting VU. Xavier Dervillez has patent #WO2017202776A1 pending to Luxembourg Institute of Health. Saskia A. Neubacher has patent #WO2019203645A1 pending to Stichting VU. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

G protein-coupled receptors (GPCRs) are pivotal therapeutic targets, but their complex structure poses challenges for effective drug design. Nanobodies, or single-domain antibodies, have emerged as a promising therapeutic strategy to target GPCRs, offering advantages over traditional small molecules and antibodies. However, an incomplete understanding of the structural features enabling GPCR-nanobody interactions has limited their development. In this study, we investigate VUN701, a nanobody antagonist targeting the atypical chemokine receptor 3 (ACKR3). We determine that an extended CDR3 loop is required for ACKR3 binding. Uncommon in most nanobodies, an extended CDR3 is prevalent in GPCR-targeting nanobodies. Combining experimental and computational approaches, we map an inhibitory ACKR3-VUN701 interface and define a distinct conformational mechanism for GPCR inactivation. Our results provide insights into class A GPCR-nanobody selectivity and suggest a strategy for the development of these new therapeutic tools., (© 2024. The Author(s).)

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5., Competing Interests: Conflict of interest E. D. and R. H. are employees of QVQ Holding BV. Other authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The therapeutic potential of ligands targeting disease-associated membrane proteins is predicted by ligand-receptor binding constants, which can be determined using NanoLuciferase (NanoLuc)-based bioluminescence resonance energy transfer (NanoBRET) methods. However, the broad applicability of these methods is hampered by the restricted availability of fluorescent probes. We describe the use of antibody fragments, like nanobodies, as universal building blocks for fluorescent probes for use in NanoBRET. Our nanobody-NanoBRET (NanoB 2 ) workflow starts with the generation of NanoLuc-tagged receptors and fluorescent nanobodies, enabling homogeneous, real-time monitoring of nanobody-receptor binding. Moreover, NanoB 2 facilitates the assessment of receptor binding of unlabeled ligands in competition binding experiments. The broad significance is illustrated by the successful application of NanoB 2 to different drug targets (e.g., multiple G protein-coupled receptors [GPCRs] and a receptor tyrosine kinase [RTK]) at distinct therapeutically relevant binding sites (i.e., extracellular and intracellular)., Competing Interests: R.H. and S.M.A. are employees of QVQ Holding B.V. R.H. and M.J.S. are inventors of patent WO2019151865A1 describing VUN100., (© 2023 The Authors.)

Introduction: The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR., Methods: Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488)., Results: These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α)., Discussion: EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes., Competing Interests: RH is CSO of QVQ Holding B.V. and SA is affiliated to QVQ Holdings. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Comez, Glenn, Anbuhl, Heukers, Smit, Hill and Kilpatrick.)

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.

The most effective treatment for HIV-1, antiretroviral therapy, suppresses viral replication and averts the disease from progression. Nonetheless, there is a need for alternative treatments as it requires daily administration with the possibility of side effects and occurrence of drug resistance. Broadly neutralizing antibodies or nanobodies targeting the HIV-1 envelope glycoprotein are explored as alternative treatment, since they mediate viral suppression and contribute to the elimination of virus-infected cells. Besides neutralization potency and breadth, Fc-mediated effector functions of bNAbs also contribute to the in vivo efficacy. In this study multivalent J3, 2E7 and 1F10 anti-HIV-1 broadly neutralizing nanobodies were generated to improve neutralization potency and IgG1 Fc fusion was utilized to gain Fc-mediated effector functions. Bivalent and trivalent nanobodies, coupled using long glycine-serine linkers, showed increased binding to the HIV-1 Env and enhanced neutralization potency compared to the monovalent variant. Fusion of an IgG1 Fc domain to J3 improved neutralization potency compared to the J3-bihead and restored Fc-mediated effector functions such as antibody-dependent cellular phagocytosis and trogocytosis, and natural killer cell activation. Due to their neutralization breadth and potency and their ability to induce effector functions these nanobody-IgG1 constructs may prove to be valuable towards alternative HIV-1 therapies., Competing Interests: Authors GD and RH were employed by QVQ Holding BV. Author TV was employed by VerLin BV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schriek, van Haaren, Poniman, Dekkers, Bentlage, Grobben, Vidarsson, Sanders, Verrips, Geijtenbeek, Heukers, Kootstra, de Taeye and van Gils.)

Photosensitizers have recently been conjugated to nanobodies for targeted photodynamic therapy (PDT) to selectively kill cancer cells. The success of this approach relies on nanobody-photosensitizer conjugates that bind specifically to their targets with very high affinities (k D in low nM range). Subsequently, upon illumination, these conjugates are very toxic and selective to cells overexpressing the target of interest (EC 50 in low nM range). In this chapter, protocols are described to determine the binding affinity of the nanobody-photosensitizer conjugates and assess the toxicity and selectivity of the conjugates when performing in vitro PDT studies. In addition, and because the efficacy of PDT also depends on the (subcellular) localization of the conjugates at the time of illumination, assays are described to investigate the uptake and the intracellular degradation of the nanobody-photosensitizer conjugates., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor's spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Latent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients., (© 2021. The Author(s).)

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gα q protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling., (© 2021. The Author(s).)

Herpesviruses are ubiquitous pathogens that establish lifelong, latent infections in their host. Spontaneous reactivation of herpesviruses is often asymptomatic or clinically manageable in healthy individuals, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with multiple proliferative cardiovascular and post-transplant diseases. Herpesviruses encode viral G protein-coupled receptors (vGPCRs) that alter the host cell by hijacking cellular pathways and play important roles in the viral life cycle and these different disease settings. In this review, we discuss the pharmacological and signaling properties of these vGPCRs, their role in the viral life cycle, and their contribution in different diseases. Because of their prominent role, vGPCRs have emerged as promising drug targets, and the potential of vGPCR-targeting therapeutics is being explored. Overall, these vGPCRs can be considered as attractive targets moving forward in the development of antiviral, cancer, and/or cardiovascular disease treatments. SIGNIFICANCE STATEMENT: In the last decade, herpesvirus-encoded G protein-coupled receptors (GPCRs) have emerged as interesting drug targets with the growing understanding of their critical role in the viral life cycle and in different disease settings. This review presents the pharmacological properties of these viral receptors, their role in the viral life cycle and different diseases, and the emergence of therapeutics targeting viral GPCRs., Competing Interests: No author has an actual or perceived conflict of interest with the contents of this article., (Copyright © 2021 by The Author(s).)

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naïve yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope.

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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15-kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognizes the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C terminus to the 11-amino-acid HiBiT tag (VUN400-HiBiT) which complements LgBiT protein, forming a full-length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect allosteric interactions with CXCR4. These data demonstrate that the high specificity offered by extracellular targeted nanobodies can be utilized to probe receptor pharmacology., Competing Interests: Declaration of Interests R.H. is CSO of QVQ Holding B.V., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gα q , Gα i , and Gα s proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies., (© 2019 van Senten et al.)

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., Competing Interests: The authors declare no conflicts of interests., (Copyright © 2019 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Photodynamic therapy (PDT) eradicates tumors by the local activation of a photosensitizer with near-infrared light. One of the aspects hampering the clinical use of PDT is the poor selectivity of the photosensitizer. To improve this, we have recently introduced a new approach for targeted PDT by conjugating photosensitizers to nanobodies. Diverse G protein-coupled receptors (GPCRs) show aberrant overexpression in tumors and are therefore interesting targets in cancer therapy. Here we show that GPCR-targeting nanobodies can be used in targeted PDT. We have developed a nanobody binding the extracellular side of the viral GPCR US28, which is detected in tumors like glioblastoma. The nanobody was site-directionally conjugated to the water-soluble photosensitizer IRDye700DX. This nanobody-photosensitizer conjugate selectively killed US28-expressing glioblastoma cells both in 2D and 3D cultures upon illumination with near-infrared light. This is the first example employing a GPCR as target for nanobody-directed PDT. With the emerging role of GPCRs in cancer, this data provides a new angle for exploiting this large family of receptors for targeted therapies.

Photodynamic therapy (PDT) is an approach that kills (cancer) cells by the local production of toxic reactive oxygen species upon the local illumination of a photosensitizer (PS). The specificity of PDT has been further enhanced by the development of a new water-soluble PS and by the specific delivery of PS via conjugation to tumor-targeting antibodies. To improve tissue penetration and shorten photosensitivity, we have recently introduced nanobodies, also known as VHH (variable domains from the heavy chain of llama heavy chain antibodies), for targeted PDT of cancer cells overexpressing the epidermal growth factor receptor (EGFR). Overexpression and activation of another cancer-related receptor, the hepatocyte growth factor receptor (HGFR, c-Met or Met) is also involved in the progression and metastasis of a large variety of malignancies. In this study we evaluate whether anti-Met VHHs conjugated to PS can also serve as a biopharmaceutical for targeted PDT. VHHs targeting the SEMA (semaphorin-like) subdomain of Met were provided with a C-terminal tag that allowed both straightforward purification from yeast supernatant and directional conjugation to the PS IRDye700DX using maleimide chemistry. The generated anti-Met VHH-PS showed nanomolar binding affinity and, upon illumination, specifically killed MKN45 cells with nanomolar potency. This study shows that Met can also serve as a membrane target for targeted PDT.

G protein-coupled receptors (GPCRs), belonging to the largest class of membrane proteins, play a prominent role in many (patho)physiological processes and are, therefore, important drug targets. Although most often targeted by small molecules, these receptors have become interesting targets for antibodies and antibody fragments, especially camelid-derived heavy chain-only antibodies and fragments thereof (nanobodies). The small size and molecular structure of nanobodies allow GPCR-binding and modulation, from both the intracellular and extracellular sides. These molecular features make nanobodies attractive tools to study, modulate, and exploit GPCRs. Besides modulating GPCR activity as monovalent or multivalent constructs, nanobodies can also be functionalized for imaging and therapy. Moreover, GPCR-binding nanobodies have been instrumental in obtaining crystal structures of GPCRs, facilitating structure-based drug discovery. Here, we describe the current status and future perspectives of nanobodies targeting GPCRs intra and extracellularly., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019. Published by Elsevier B.V.)

Antibody-based pharmaceuticals often encompass a complex structural heterogeneity requiring enhanced analytical methods for reliable characterization of variants and degradation products. We have explored the capabilities of low-flow sheathless capillary electrophoresis-mass spectrometry (CE-MS) for the high-resolution and sensitive profiling of antibody therapeutics. Near-zero electroosmotic flow was achieved by employing a novel neutral capillary coating that also prevents protein adsorption. CE-MS analysis of intact model proteins using an acidic background electrolyte demonstrated satisfactory performance, with overall migration-time RSDs below 2.2% from three different capillaries tested. For system evaluation, three nanobody preparations, including mono- and bivalent forms, and three monoclonal antibodies (mAbs) were analyzed. Intact nanobodies were resolved from their degradation products, which could be assigned to deamidated, cleaved, and truncated forms at the C-terminal tag. Excellent resolution of isomeric deamidated products was obtained. The mAbs were analyzed intact and after digestion by the endoproteinase IdeS (middle-up approach). CE-MS of intact mAbs provided resolution of clipped species (e.g. light chain and light chain-heavy chain fragments) from the native protein. Moreover, glycoforms containing sialic acids were resolved from their non-sialylated counterparts. For IdeS-digested, F (ab) 2 and Fc/2 portions where efficiently resolved for the three mAbs. Whereas the migration time of the Fc/2 fragments was fairly similar, the migration time of the F (ab) 2 part was strongly varied among the mAbs. For all mAbs, separation of Fc/2 charge variants - including sialylated glycoforms and other post-translational modifications, such as loss of C-terminal lysine or asparagine deamidation - was achieved. This allowed a detailed and reliable assessment of the Fc/2 heterogeneity (18-33 proteoforms) of the three analyzed mAbs., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

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., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. K IR 2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (I K 1 ), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in K IR 2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited I K 1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased K IR 2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 μM, 24 hrs) and late-endosomal/lysosomal K IR 2.1 accumulation. Increased K IR 2.1 expression level was also observed in the presence of Na v 1.5 co-expression. Augmented K IR 2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on K v 11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at -120 mV, 5 μM) enhanced I KIR 2.1 upon 24-hrs treatment, whereas dronedarone tended to increase I KIR 2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at -120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced I K 1 by inhibiting K IR 2.1 degradation., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer (PS) and has been used to treat head and neck cancers. Yet, common PS lack tumor specificity, which leads to collateral damage to normal tissues. Targeted delivery of PS via antibodies has pre-clinically improved tumor selectivity. However, antibodies have long half-lives and relatively poor tissue penetration, which could limit therapeutic efficacy and lead to long photosensitivity. Here, in this feasibility study, we evaluate at the pre-clinical level a recently introduced format of targeted PDT, which employs nanobodies as targeting agents and a water-soluble PS (IRDye700DX) that is traceable through optical imaging. In vitro, the PS solely binds to cells and induces phototoxicity on cells overexpressing the epidermal growth factor receptor (EGFR), when conjugated to the EGFR targeted nanobodies. To investigate whether this new format of targeted PDT is capable of inducing selective tumor cell death in vivo, PDT was applied on an orthotopic mouse tumor model with illumination at 1h post-injection of the nanobody-PS conjugates, as selected from quantitative fluorescence spectroscopy measurements. In parallel, and as a reference, PDT was applied with an antibody-PS conjugate, with illumination performed 24h post-injection. Importantly, EGFR targeted nanobody-PS conjugates led to extensive tumor necrosis (approx. 90%) and almost no toxicity in healthy tissues, as observed through histology 24h after PDT. Overall, results show that these EGFR targeted nanobody-PS conjugates are selective and able to induce tumor cell death in vivo. Additional studies are now needed to assess the full potential of this approach to improving PDT., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies., (©2015 American Association for Cancer Research.)

Photodynamic therapy (PDT) induces cell death through light activation of a photosensitizer (PS). Targeted delivery of PS via monoclonal antibodies has improved tumor selectivity. However, these conjugates have long half-lives, leading to relatively long photosensitivity in patients. In an attempt to target PS specifically to tumors and to accelerate PS clearance, we have developed new conjugates consisting of nanobodies (NB) targeting the epidermal growth factor receptor (EGFR) and a traceable PS (IRDye700DX). These fluorescent conjugates allow the distinction of cell lines with different expression levels of EGFR. Results show that these conjugates specifically induce cell death of EGFR overexpressing cells in low nanomolar concentrations, while PS alone or the NB-PS conjugates in the absence of light induce no toxicity. Delivery of PS using internalizing biparatopic NB-PS conjugates results in even more pronounced phototoxicities. Altogether, EGFR-targeted NB-PS conjugates are specific and potent, enabling the combination of molecular imaging with cancer therapy. From the clinical editor: This study investigates the role of EGFR targeting nanobodies to deliver traceable photosensitizers to cancer molecules for therapeutic exploitation and concomitant imaging. Altogether, EGFR-targeted NB-PS conjugates combine molecular imaging with cancer therapy, the method is specific and potent, paving the way to clinical application of this technology., (Copyright © 2014 Elsevier Inc. All rights reserved.)