Bibliometric and scientometric analysis of PSMA-targeted radiotheranostics: knowledge mapping and global standing.

Purpose: Bibliometric and scientometric analyses provide a structured approach to large amounts of data, enabling the prediction of research theme trends over time, the detection of shifts in the boundaries of disciplines, and the identification of the most productive countries, institutions and scholars. In the context of prostate-specific membrane antigen (PSMA)-targeted radiotheranostics, no bibliometric or scientometric analysis has been published thus far. Therefore, this study was conducted to identify key contributors to the literature, assess the global scientific production of related research, and possibly predict future development patterns. Methods: Scientometrics and bibliometrics were utilized to analyze the current body of knowledge while tracking its evolution to support scientific decision-making comprehensively and systematically. Science mapping techniques were employed to visualize research activities. Two different tools, Tableau and VOSviewer, were utilized, with VOSviewer being deemed the most suitable for the research objectives. The Web of Science (WoS) was used as the principal database for the searches. Results: Through the search process over a period of 30 years (January 1993-January 2023), 694 original studies in the English language were subjected to comprehensive analysis. By employing bibliometric and scientometric methods, multiple networks were created that mapped various concepts, such as publication trends, leading countries, cocitations, coauthorship among researchers and scientists, as well as coauthorship among organizations and funding agencies. This study revealed the evolutionary patterns, trends, outliers, and key players in the PSMA field, which enabled a more nuanced understanding of the research landscape. Conclusion: This research contributes to the enrichment of knowledge on PSMA-targeted radiotheranostics through detailed global bibliometric and scientometric analyses. It stresses the necessity for the development of communication platforms, the establishment of supportive infrastructures, and the implementation of proactive solutions to address emerging challenges. This study offers a significant resource for delineating effective strategies and identifying prominent funding bodies essential for continuous advancements in the field of PSMA-based diagnosis and therapy for prostate cancer. It is vital to sustain this momentum to ensure further progress in this pioneering area.

PSMA-targeted radiotheranostics in modern nuclear medicine: then, now, and what of the future?

In 1853, the perception of prostate cancer (PCa) as a rare ailment prevailed, was described by the eminent Londoner surgeon John Adams. Rapidly forward to 2018, the landscape dramatically altered. Currently, men face a one-in-nine lifetime risk of PCa, accentuated by improved diagnostic methods and an ageing population. With more than three million men in the United States alone grappling with this disease, the overall risk of succumbing to stands at one in 39. The intricate clinical and biological diversity of PCa poses serious challenges in terms of imaging, ongoing monitoring, and disease management. In the field of theranostics, diagnostic and therapeutic approaches that harmoniously merge targeted imaging with treatments are integrated. A pivotal player in this arena is radiotheranostics, employing radionuclides for both imaging and therapy, with prostate-specific membrane antigen (PSMA) at the forefront. Clinical milestones have been reached, including FDA- and/or EMA-approved PSMA-targeted radiodiagnostic agents, such as [18F]DCFPyL (PYLARIFY®, Lantheus Holdings), [18F]rhPSMA-7.3 (POSLUMA®, Blue Earth Diagnostics) and [68Ga]Ga-PSMA-11 (Locametz®, Novartis/ ILLUCCIX®, Telix Pharmaceuticals), as well as PSMA-targeted radiotherapeutic agents, such as [177Lu]Lu-PSMA-617 (Pluvicto®, Novartis). Concurrently, ligand-drug and immune therapies designed to target PSMA are being advanced through rigorous preclinical research and clinical trials. This review delves into the annals of PSMA-targeted radiotheranostics, exploring its historical evolution as a signature molecule in PCa management. We scrutinise its clinical ramifications, acknowledge its limitations, and peer into the avenues that need further exploration. In the crucible of scientific inquiry, we aim to illuminate the path toward a future where the enigma of PCa is deciphered and where its menace is met with precise and effective countermeasures. In the following sections, we discuss the intriguing terrain of PCa radiotheranostics through the lens of PSMA, with the fervent hope of advancing our understanding and enhancing clinical practice.

Nanobody-based pannexin1 channel inhibitors reduce inflammation in acute liver injury.

BACKGROUND: The opening of pannexin1 channels is considered as a key event in inflammation. Pannexin1 channel-mediated release of adenosine triphosphate triggers inflammasome signaling and activation of immune cells. By doing so, pannexin1 channels play an important role in several inflammatory diseases. Although pannexin1 channel inhibition could represent a novel clinical strategy for treatment of inflammatory disorders, therapeutic pannexin1 channel targeting is impeded by the lack of specific, potent and/or in vivo-applicable inhibitors. The goal of this study is to generate nanobody-based inhibitors of pannexin1 channels. RESULTS: Pannexin1-targeting nanobodies were developed as potential new pannexin1 channel inhibitors. We identified 3 cross-reactive nanobodies that showed affinity for both murine and human pannexin1 proteins. Flow cytometry experiments revealed binding capacities in the nanomolar range. Moreover, the pannexin1-targeting nanobodies were found to block pannexin1 channel-mediated release of adenosine triphosphate. The pannexin1-targeting nanobodies were also demonstrated to display anti-inflammatory effects in vitro through reduction of interleukin 1 beta amounts. This anti-inflammatory outcome was reproduced in vivo using a human-relevant mouse model of acute liver disease relying on acetaminophen overdosing. More specifically, the pannexin1-targeting nanobodies lowered serum levels of inflammatory cytokines and diminished liver damage. These effects were linked with alteration of the expression of several NLRP3 inflammasome components. CONCLUSIONS: This study introduced for the first time specific, potent and in vivo-applicable nanobody-based inhibitors of pannexin1 channels. As demonstrated for the case of liver disease, the pannexin1-targeting nanobodies hold great promise as anti-inflammatory agents, yet this should be further tested for extrahepatic inflammatory disorders. Moreover, the pannexin1-targeting nanobodies represent novel tools for fundamental research regarding the role of pannexin1 channels in pathological and physiological processes.

Screening and epitope characterization of diagnostic nanobody against total and activated Bacteroides fragilis toxin.

Enterotoxigenic Bacteroides fragilis (ETBF) can rapidly secrete an enterotoxin termed B. fragilis toxin (BFT), which is thought to be the only recognized virulence factor in ETBF. ETBF can cause acute diarrhea, inflammatory bowel disease (IBD), colorectal cancer, and breast cancer. BFT is divided into three subtypes, BFT1, BFT2, and BFT3. BFT1 is the most widely distributed in human B. fragilis isolates. BFT can be used as a biomarker for predicting the inflammation-cancer transformation of intestine and breast. Nanobodies have the advantages of small structure, complete antigen recognition capacity, rapid selection via phage display technology, and can be massively produced in microbial expression systems. Nanobodies have become a powerful tool for medical diagnosis and treatment. This study focuses on screening and structural characterization of nanobodies targeting full length and active BFT. By constructing prokaryotic expression systems to obtain recombinant BFT1 protein, high purity BFT1 protein was used to immunize alpacas. Phage display technology was used to construct a phage display library. The positive clones were selected by bio-panning, and the isothermal titration calorimetry was used to select high-affinity nanobodies. Then the three-dimensional structures of BFT1:Nb2.82 and BFT1:Nb3.27 were solved by crystal X-ray diffraction. We got two kinds of nanobodies, Nb2.82 targeting the BFT1 prodomain and Nb3.27 recognizing the BFT1 catalytic domain. This study provides a new strategy for the early diagnosis of ETBF and the possibility for BFT as a biomarker for diagnosing diseases.

Nanobodies targeting ABCC3 for immunotargeted applications in glioblastoma.

The cancer "omics" reveal many clinically relevant alterations that are transforming the molecular characterization of glioblastomas. However, many of these findings are not yet translated into clinical practice due, in part, to the lack of non-invasive biomarkers and the limitations imposed by the blood-brain barrier. Nanobodies, camelid single-domain antibody fragments, emerge as a promising tool for immunotargeted applications for diagnosing and treating glioblastomas. Performing agnostic bioinformatic analysis from glioblastoma patient datasets, we identified ATP Binding Cassette subfamily C member 3 (ABCC3) as a suitable target for immunotargeted applications. The expression of ABCC3 is associated with poor survival and impaired response to temozolomide. Importantly, high expression of ABCC3 is restricted to glioblastoma, with negligible levels in healthy brain tissue, and further correlates with tumor grade and stemness markers. We identified three immunogenic epitopes of ABCC3 which were used to isolate nanobodies from a glioblastoma-specific phage-display nanobody library. Two nanobodies targeting ABCC3 (NbA42 and NbA213) were further characterized and demonstrated in vivo selective recognition of ABCC3 in glioblastoma xenograft mouse models upon systemic administration. We designate NbA42 and NbA213 as new candidates to implement immunotargeted applications guiding a more personalized and precise diagnosis, monitoring, and treatment of glioblastoma patients.

Nanobodies for the Early Detection of Ovarian Cancer.

Ovarian cancer ranks fifth in cancer-related deaths among women. Since ovarian cancer patients are often asymptomatic, most patients are diagnosed only at an advanced stage of disease. This results in a 5-year survival rate below 50%, which is in strong contrast to a survival rate as high as 94% if detected and treated at an early stage. Monitoring serum biomarkers offers new possibilities to diagnose ovarian cancer at an early stage. In this study, nanobodies targeting the ovarian cancer biomarkers human epididymis protein 4 (HE4), secretory leukocyte protease inhibitor (SLPI), and progranulin (PGRN) were evaluated regarding their expression levels in bacterial systems, epitope binning, and antigen-binding affinity by enzyme-linked immunosorbent assay and surface plasmon resonance. The selected nanobodies possess strong binding affinities for their cognate antigens (KD~0.1-10 nM) and therefore have a pronounced potential to detect ovarian cancer at an early stage. Moreover, it is of utmost importance that the limits of detection (LOD) for these biomarkers are in the pM range, implying high specificity and sensitivity, as demonstrated by values in human serum of 37 pM for HE4, 163 pM for SLPI, and 195 pM for PGRN. These nanobody candidates could thus pave the way towards multiplexed biosensors.

Identification of Serum Ferritin-Specific Nanobodies and Development towards a Diagnostic Immunoassay.

Serum ferritin (SF) is an iron-rich protein tightly connected with iron homeostasis, and the variations are frequently observed in diseased states, including iron-deficiency anemia, inflammation, liver disease, and tumors, which renders SF level an indicator of potential malignancies in clinical practice. Nanobodies (Nbs) have been widely explored and developed into theranostic reagents. Surprisingly, no reports stated the identification of anti-SF Nbs, nor the potential of such Nbs as a diagnostic tool. In this study, we generated SF-specific Nbs and provided novel clinical diagnostic approaches to develop an immunoassay. An immune library was constructed after immunizing an alpaca with SF, and five Nbs specifically targeting human SF were retrieved. The obtained Nbs exhibited robust properties including high stability, affinity, and specificity. Then, an ELISA-based test using a heterologous Nb-pair was developed. The calibration curve demonstrated a linear range of SF between 9.0 to 1100 ng/mL, and a limit of detection (LOD) of 1.01 ng/mL. The detecting recovery and coefficient variation (CV) were determined by spiking different concentrations of SF into the serum sample, to verify the successful application of our selected Nbs for SF monitoring. In general, this study generated SF-specific Nbs and demonstrated their potential as diagnostic immunoassay tools.

Sensitive Protein Detection Using Site-Specifically Oligonucleotide-Conjugated Nanobodies.

High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.

An overview on display systems (phage, bacterial, and yeast display) for production of anticancer antibodies; advantages and disadvantages.

Antibodies as ideal therapeutic and diagnostic molecules are among the top-selling drugs providing considerable efficacy in disease treatment, especially in cancer therapy. Limitations of the hybridoma technology as routine antibody generation method in conjunction with numerous developments in molecular biology led to the development of alternative approaches for the streamlined identification of most effective antibodies. In this regard, display selection technologies such as phage display, bacterial display, and yeast display have been widely promoted over the past three decades as ideal alternatives to traditional methods. The display of antibodies on phages is probably the most widespread of these methods, although surface display on bacteria or yeast have been employed successfully, as well. These methods using various sizes of combinatorial antibody libraries and different selection strategies possessing benefits in screening potency, generating, and isolation of high affinity antibodies with low risk of immunogenicity. Knowing the basics of each method assists in the design and retrieval process of antibodies suitable for different diseases, including cancer. In this review, we aim to outline the basics of each library construction and its display method, screening and selection steps. The advantages and disadvantages in comparison to alternative methods, and their applications in antibody engineering will be explained. Finally, we will review approved or non-approved therapeutic antibodies developed by employing these methods, which may serve as therapeutic antibodies in cancer therapy.

AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model.

Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL-G-F Alzheimer's mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

Cytoplasmic Expression of Nanobodies with Formylglycine Generating Enzyme Tag and Conversion to a Bio-Orthogonal Aldehyde Group.

Nanobodies (Nbs) can be successfully retrieved following phage, bacterial, yeast, or ribosome display of immune, synthetic, or naïve libraries. However, after panning, multiple individual Nb clones need to be screened and assessed for solubility, antigen specificity, affinity, and potential biological function. Therefore, it is highly desirable to have a convenient expression strategy to obtain sufficient protein for in-depth characterization of the Nbs. The presence of a purification and detection tag, as well as a chemically reactive group to enable simple generation of Nb derivatives, would be of great help in this regard. Here, we provide a general protocol for high yield cytoplasmic expression and purification of formylglycine generating enzyme (FGE)-tagged Nbs. The cysteine within the FGE tag is easily converted to formylglycine by passing the FGE-tag containing Nb over a continuous-flow bio-catalysis system. The aldehyde group within the formylglycine side chain at the C-terminal end of the Nb is suitably located for subsequent bio-orthogonal reactions to fluorescent dyes, biotin, polyethylene glycol, or chromatography resins. We also include methods for production of high yield recombinant FGE, as well as conditions for its immobilization on Sepharose to produce the continuous-flow bio-catalysis system.

Imaging of Glioblastoma Tumor-Associated Myeloid Cells Using Nanobodies Targeting Signal Regulatory Protein Alpha.

Glioblastoma (GBM) is the most common malignant primary brain tumor. Glioblastomas contain a large non-cancerous stromal compartment including various populations of tumor-associated macrophages and other myeloid cells, of which the presence was documented to correlate with malignancy and reduced survival. Via single-cell RNA sequencing of human GBM samples, only very low expression of PD-1, PD-L1 or PD-L2 could be detected, whereas the tumor micro-environment featured a marked expression of signal regulatory protein alpha (SIRPα), an inhibitory receptor present on myeloid cells, as well as its widely distributed counter-receptor CD47. CITE-Seq revealed that both SIRPα RNA and protein are prominently expressed on various populations of myeloid cells in GBM tumors, including both microglia- and monocyte-derived tumor-associated macrophages (TAMs). Similar findings were obtained in the mouse orthotopic GL261 GBM model, indicating that SIRPα is a potential target on GBM TAMs in mouse and human. A set of nanobodies, single-domain antibody fragments derived from camelid heavy chain-only antibodies, was generated against recombinant SIRPα and characterized in terms of affinity for the recombinant antigen and binding specificity on cells. Three selected nanobodies binding to mouse SIRPα were radiolabeled with 99mTc, injected in GL261 tumor-bearing mice and their biodistribution was evaluated using SPECT/CT imaging and radioactivity detection in dissected organs. Among these, Nb15 showed clear accumulation in peripheral organs such as spleen and liver, as well as a clear tumor uptake in comparison to a control non-targeting nanobody. A bivalent construct of Nb15 exhibited an increased accumulation in highly vascularized organs that express the target, such as spleen and liver, as compared to the monovalent format. However, penetration into the GL261 brain tumor fell back to levels detected with a non-targeting control nanobody. These results highlight the tumor penetration advantages of the small monovalent nanobody format and provide a qualitative proof-of-concept for using SIRPα-targeting nanobodies to noninvasively image myeloid cells in intracranial GBM tumors with high signal-to-noise ratios, even without blood-brain barrier permeabilization.

CS1-specific single-domain antibodies labeled with Actinium-225 prolong survival and increase CD8+ T cells and PD-L1 expression in Multiple Myeloma.

Multiple myeloma (MM) is a hematological malignancy characterized by the presence of clonal plasma cells in the bone marrow niche. Despite significant therapeutic advances, MM remains incurable for the majority of patients. Targeted radionuclide therapy (TRNT) has emerged as a promising treatment option to eradicate residual cancer cells. In this study, we developed and characterized single-domain antibodies (sdAbs) against the MM-antigen CS1 and evaluated its therapeutic potential in MM using TRNT. We first validated CS1 as potential target for TRNT. CS1 is expressed in normal and malignant plasma cells in different disease stages including progression and relapse. It is expressed in dormant as well as proliferating MM cells, while low expression could be observed in environmental cells. Biodistribution studies demonstrated the specific uptake of anti-CS1 sdAbs in tissues of 5TMM cell infiltration including bone, spleen and liver. TRNT using anti-CS1 sdAbs labeled with actinium-225 significantly prolonged survival of syngeneic, immunocompetent 5T33MM mice. In addition, we observed an increase in CD8+ T-cells and more overall PD-L1 expression on immune and non-immune cells, implying an interferon gamma signature using actinium-225 labeled CS1-directed sdAbs. In this proof-of-principle study, we highlight, for the first time, the therapeutic potential and immunomodulating effects of anti-CS1 radionuclide therapy to target residual MM cells.

Intrabody Targeting HIF-1α Mediates Transcriptional Downregulation of Target Genes Related to Solid Tumors.

Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.

Direct Immobilization of Engineered Nanobodies on Gold Sensors.

Single-domain antibodies, known as nanobodies, have great potential as biorecognition elements for sensors because of their small size, affinity, specificity, and robustness. However, facile and efficient methods of nanobody immobilization are sought that retain their maximum functionality. Herein, we describe the direct immobilization of nanobodies on gold sensors by exploiting a modified cysteine strategically positioned at the C-terminal end of the nanobody. The experimental data based on secondary ion mass spectrometry, circular dichroism, and surface plasmon resonance, taken together with a detailed computational work (molecular dynamics simulations), support the formation of stable and well-oriented nanobody monolayers. Furthermore, the nanobody structure and activity is preserved, wherein the nanobody is immobilized at a high density (approximately 1 nanobody per 13 nm2). The strategy for the spontaneous nanobody self-assembly is simple and effective and possesses exceptional potential to be used in numerous sensing platforms, ranging from clinical diagnosis to environmental monitoring.

Mechanisms Underlying Connexin Hemichannel Activation in Disease.

Gap junctions and connexin hemichannels mediate intercellular and extracellular communication, respectively. While gap junctions are seen as the "good guys" by controlling homeostasis, connexin hemichannels are considered as the "bad guys", as their activation is associated with the onset and dissemination of disease. Open connexin hemichannels indeed mediate the transport of messengers between the cytosol and extracellular environment and, by doing so, fuel inflammation and cell death in a plethora of diseases. The present mini-review discusses the mechanisms involved in the activation of connexin hemichannels during pathology.

Immunogenicity Risk Profile of Nanobodies.

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications. One Sentence Summary: Nanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor.

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters.

Nanobodies against the metal binding domains of ATP7B as tools to study copper transport in the cell.

Nanobodies are genetically engineered single domain antibodies derived from the unusual heavy-chain only antibodies found in llamas and camels. The small size of the nanobodies and flexible selection schemes make them uniquely versatile tools for protein biochemistry and cell biology. We have developed a panel of nanobodies against the metal binding domains of the human copper transporter ATP7B, a multidomain membrane protein with a complex regulation of enzymatic activity and intracellular localization. To enable the use of the nanobodies as tools to investigate copper transport in the cell, we characterized their binding sites and affinity by isothermal titration calorimetry and NMR. We have identified nanobodies against each of the first four metal binding domains of ATP7B, with a wide affinity range, as evidenced by dissociation constants from below 10-9 to 10-6 M. We found both the inhibitory and activating nanobodies among those tested. The diverse properties of the nanobodies make the panel useful for the structural studies of ATP7B, immunoaffinity purification of the protein, modulation of its activity in the cell, protein dynamics studies, and as mimics of copper chaperone ATOX1, the natural interaction partner of ATP7B.

Structural basis of nanobody recognition of grapevine fanleaf virus and of virus resistance loss.

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.