Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica).

Diverse candidate antibodies are needed to successfully identify therapeutic and diagnostic applications. The variable domain of IgNAR (VNAR), a shark single-domain antibody, has attracted attention owing to its favorable physicochemical properties. The phage display method used to screen for optimal VNARs loses sequence diversity because of the bias caused by the differential ease of protein expression in Escherichia coli. Here, we investigated a VNAR selection method that combined panning with various selection pressures and next-generation sequencing (NGS) analyses to obtain additional candidates. Drawing inspiration from the physiological conditions of sharks and the physicochemical properties of VNARs, we examined the effects of NaCl and urea concentrations, low temperature, and preheating at the binding step of panning. VNAR phage libraries generated from Japanese topeshark (Hemitriakis japanica) were enriched under these conditions. We then performed NGS analysis and attempted to select clones that were specifically enriched under each panning condition. The identified VNARs exhibited higher reactivity than those obtained by panning without selection pressure. Additionally, they possess physicochemical properties that reflect their respective selection pressures. These results can greatly enhance our understanding of VNAR properties and offer guidance for the screening of high-quality VNAR clones that are present at low frequencies.

Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation.

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase.

The soluble epoxide hydrolase (sEH) is possibly both a marker for and target of numerous diseases. Herein, we describe a homogeneous mix-and-read assay for the detection of human sEH based on using split-luciferase detection coupled with anti-sEH nanobodies. Selective anti-sEH nanobodies were individually fused with NanoLuc Binary Technology (NanoBiT), which consists of a large and small portion of NanoLuc (LgBiT and SmBiT, respectively). Different orientations of the LgBiT and SmBiT-nanobody fusions were expressed and investigated for their ability to reform the active NanoLuc in the presence of the sEH. After optimization, the linear range of the assay could reach 3 orders of magnitude with a limit of detection (LOD) of 1.4 ng/mL. The assay has a high sensitivity to human sEH and reached a similar detection limit to our previously reported conventional nanobody-based ELISA. The procedure of the assay was faster (30 min total) and easy to operate, providing a more flexible and simple way to monitor human sEH levels in biological samples. In general, the immunoassay proposed here offers a more efficient detection and quantification approach that can be easily adapted to numerous macromolecules.

Antibody-based defense may protect plants from disease.

A "creative" strategy to keep crops healthy borrows key pathogen detectors from the animal immune system.

NLR immune receptor-nanobody fusions confer plant disease resistance.

Plant pathogens cause recurrent epidemics, threatening crop yield and global food security. Efforts to retool the plant immune system have been limited to modifying natural components and can be nullified by the emergence of new pathogen strains. Made-to-order synthetic plant immune receptors provide an opportunity to tailor resistance to pathogen genotypes present in the field. In this work, we show that plant nucleotide-binding, leucine-rich repeat immune receptors (NLRs) can be used as scaffolds for nanobody (single-domain antibody fragment) fusions that bind fluorescent proteins (FPs). These fusions trigger immune responses in the presence of the corresponding FP and confer resistance against plant viruses expressing FPs. Because nanobodies can be raised against most molecules, immune receptor-nanobody fusions have the potential to generate resistance against plant pathogens and pests delivering effectors inside host cells.

Nebulized delivery of a broadly neutralizing SARS-CoV-2 RBD-specific nanobody prevents clinical, virological, and pathological disease in a Syrian hamster model of COVID-19.

There remains an unmet need for globally deployable, low-cost therapeutics for the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Previously, we reported on the isolation and in vitro characterization of a potent single-domain nanobody, NIH-CoVnb-112, specific for the receptor-binding domain (RBD) of SARS-CoV-2. Here, we report on the molecular basis for the observed broad in vitro neutralization capability of NIH-CoVnb-112 against variant SARS-CoV-2 pseudoviruses. The structure of NIH-CoVnb-112 bound to SARS-CoV-2 RBD reveals a large contact surface area overlapping the angiotensin converting enzyme 2 (ACE2) binding site, which is largely unencumbered by the common RBD mutations. In an in vivo pilot study, we demonstrate effective reductions in weight loss, viral burden, and lung pathology in a Syrian hamster model of COVID-19 following nebulized delivery of NIH-CoVnb-112. These findings support the further development of NIH-CoVnb-112 as a potential adjunct preventative therapeutic for the treatment of SARS-CoV-2 infection.Abbreviations: ACE2 - angiotensin converting enzyme 2BSA - buried surface areaCDR - complementary determining regionRBD - receptor binding domainRBM - receptor-binding motifSARS-CoV-2 - severe acute respiratory syndrome coronavirus 2.

First-in-class trispecific VHH-Fc based antibody with potent prophylactic and therapeutic efficacy against SARS-CoV-2 and variants.

SARS-CoV-2 and its variants have persisted in this ongoing COVID-19 pandemic. While the vaccines have greatly reduced the COVID-19 cases, hospitalizations, and death, about half of the world remain unvaccinated due to various reasons. Furthermore, the duration of the immunity gained from COVID-19 vaccination is still unclear. Therefore, there is a need for innovative prophylactic and treatment measures. In response to this need, we previously reported on the successful computer-aided development of potent VHH-based multispecific antibodies that were characterized in vitro. Here, we evaluated in vivo efficacy and safety of the lead trispecific VHH-Fc, ABS-VIR-001. Importantly, our data showed that ABS-VIR-001 treatment prevented SARS-CoV-2 infection and death when provided as an intranasal prophylaxis in a humanized ACE-2 mouse model. In addition, ABS-VIR-001 post-exposure treatment was shown to greatly reduce viral loads by as much as 50-fold. A detailed panel of metabolic and cellular parameters demonstrated that ABS-VIR-001 treatment was overall comparable to the PBS treatment, indicating a favorable safety profile. Notably, our inhibition studies show that ABS-VIR-001 continued to demonstrate unwavering efficacy against SARS-CoV-2 mutants, associated with key variants including Delta and Omicron, owing to its multiple epitope design. Lastly, we rigorously tested and confirmed the excellent thermostability of ABS-VIR-001 when heated to 45 °C for up to 4 weeks. Taken together, our study suggests that ABS-VIR-001 is an efficacious and durable prophylaxis and post-exposure treatment for COVID-19 with promising safety and manufacturability features for global distribution.

Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools.

Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research.

Generation of nanobodies targeting the human, transcobalamin-mediated vitamin B12 uptake route.

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B12 ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of these nanobodies bound to holo-TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC-Nb4) using fluorescent light microscopy. The co-crystal structure of holo-TC:TCblR with another nanobody (TC-Nb34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR, rationalizing the decrease in the affinity of TC-B12 binding caused by the Δ88 mutation in CD320.

Outlook of therapeutic and diagnostic competency of nanobodies against SARS-CoV-2: A systematic review.

PURPOSE: The newly emerged coronavirus (SARS-CoV-2) continues to infect humans, and no completely efficient treatment has yet been found. Antibody therapy is one way to control infection caused by COVID-19, but the use of classical antibodies has many disadvantages. Heavy chain antibodies (HCAbs) are single-domain antibodies derived from the Camelidae family. The variable part of these antibodies (Nanobodies or VHH) has interesting properties such as small size, identify criptic epitopes, stability in harsh conditions, good tissue permeability and cost-effective production causing nanobodies have become a good candidate in the treatment and diagnosis of viral infections. METHODS: Totally 157 records (up to November 10, 2021), were recognized to be reviewed in this study. 62 studies were removed after first step screening due to their deviation from inclusion criteria. The remaining 95 studies were reviewed in details. After removing articles that were not in the study area, 45 remaining studies met the inclusion criteria and were qualified to be included in the systematic review. RESULTS: In this systematic review, the application of nanobodies in the treatment and detection of COVID-19 infection was reviewed. The results of this study showed that extensive and sufficient studies have been performed in the field of production of nanobodies against SARS-CoV-2 virus and the obtained nanobodies have a great potential for use in patients infected with SARS-CoV-2 virus. CONCLUSION: According to the obtained results, it was found that nanobodies can be used effectively in the treatment and diagnosis of SARS-CoV-2 virus.

SAbDab in the age of biotherapeutics: updates including SAbDab-nano, the nanobody structure tracker.

In 2013, we released the Structural Antibody Database (SAbDab), a publicly available repository of experimentally determined antibody structures. In the interim, the rapid increase in the number of antibody structure depositions to the Protein Data Bank, driven primarily by increased interest in antibodies as biotherapeutics, has led us to implement several improvements to the original database infrastructure. These include the development of SAbDab-nano, a sub-database that tracks nanobodies (heavy chain-only antibodies) which have seen a particular growth in attention from both the academic and pharmaceutical research communities over the past few years. Both SAbDab and SAbDab-nano are updated weekly, comprehensively annotated with the latest features described here, and are freely accessible at opig.stats.ox.ac.uk/webapps/newsabdab/.

A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo.

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants.

Rapid discovery of diverse neutralizing SARS-CoV-2 antibodies from large-scale synthetic phage libraries.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

Llamanade: An open-source computational pipeline for robust nanobody humanization.

Nanobodies (Nbs) have emerged as a promising class of biologics. Despite having marked physicochemical properties, Nbs are derived from camelids and may require humanization to improve translational potentials. By systematically analyzing the sequence and structural properties of Nbs, we found substantial framework diversities and revealed the key differences between Nbs and human immunoglobulin G antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade," an open-source software to facilitate rational humanization of Nbs. Using sequence as input, Llamanade can rapidly extract sequence features, model structures, and optimize solutions to humanize Nbs. Finally, we used Llamanade to successfully humanize a cohort of structurally diverse and potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a server to support the development of therapeutic Nbs into safe and effective trials.

INDI-integrated nanobody database for immunoinformatics.

Nanobodies, a subclass of antibodies found in camelids, are versatile molecular binding scaffolds composed of a single polypeptide chain. The small size of nanobodies bestows multiple therapeutic advantages (stability, tumor penetration) with the first therapeutic approval in 2018 cementing the clinical viability of this format. Structured data and sequence information of nanobodies will enable the accelerated clinical development of nanobody-based therapeutics. Though the nanobody sequence and structure data are deposited in the public domain at an accelerating pace, the heterogeneity of sources and lack of standardization hampers reliable harvesting of nanobody information. We address this issue by creating the Integrated Database of Nanobodies for Immunoinformatics (INDI, http://naturalantibody.com/nanobodies). INDI collates nanobodies from all the major public outlets of biological sequences: patents, GenBank, next-generation sequencing repositories, structures and scientific publications. We equip INDI with powerful nanobody-specific sequence and text search facilitating access to >11 million nanobody sequences. INDI should facilitate development of novel nanobody-specific computational protocols helping to deliver on the therapeutic promise of this drug format.

Single-domain antibody screening by isPLA-seq.

Single-domain antibody (sdAb) holds the promising strategies for diverse research and translational applications. Here, we describe a method for the adaptation of the in situ proximity ligation assay (isPLA) followed by sequencing (isPLA-seq) to facilitate screening of a high-sensitive, high-throughput sdAb library for a given protein at subcellular and single-cell resolution. Based on the sequence of complementarity-determining region 3 (CDR3), the recombinant sdAb can be produced for in vitro and in vivo utilities. This method provides a general means to identify the functional measure of sdAb and its complementary epitopes and its potential applications to investigate cellular processes.

Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification.

Nanobodies (Nbs) have shown great potential in immunodetection of small-molecule contaminants in food and environmental monitoring. However, the limited knowledge of the mechanism of Nbs binding to small molecules has hampered the development of high-affinity Nbs and assay improvement. We previously reported two homologous nanobodies Nb26 and Nb28 specific to aflatoxin B1 (AFB1), with the former exhibiting higher sensitivity in ELISA. Herein, Nb26 was selected as the model antibody to resolve its solution nuclear magnetic resonance (NMR) structure, and investigate its AFB1 recognition mechanism. The results revealed that Nb26 exhibits a typical immunoglobulin fold and its AFB1-binding interface is uniquely located in complementarity-determining region 3 (CDR3) and framework region 2 (FR2). This finding was applied to improve the binding activity of Nb28 against AFB1 by constructing two Nb28-based mutants A50V and S102D, resulting in 2.3- and 3.3-fold sensitivity enhancement over the wild type, respectively. This study develops an NMR-based strategy to analyze the underlying mechanism of Nb against AFB1, and successfully generated two site-modified Nbs with improved detection sensitivity. It is believed that this work could greatly expand the applications of Nbs by providing a way to enhance the binding activity.

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

The role of single-domain antibodies (or nanobodies) in SARS-CoV-2 neutralization.

The severe acute respiratory syndrome (SARS-CoV-2), a newly emerging of coronavirus, continues to infect humans in the absence of a viable treatment. Neutralizing antibodies that disrupt the interaction of RBD and ACE2 has been under the spotlight as a way of developing the COVID-19 treatment. Some animals, such as llamas, manufacture heavy-chain antibodies that have a single variable domain (VHH) instead of two variable domains (VH/VL) as opposed to typical antibodies. Nanobodies are antigen-specific, single-domain, changeable segments of camelid heavy chain-only antibodies that are recombinantly produced. These types of antibodies exhibit a wide range of strong physical and chemical properties, like high solubility, and stability. The VHH's high-affinity attachment to the receptor-binding domain (RBD) allowed the neutralization of SARS-CoV-2. To tackle COVID-19, some nanobodies are being developed against SARS-CoV-2, some of which have been recently included in clinical trials. Nanobody therapy may be useful in managing the COVID-19 pandemic as a potent and low-cost treatment. This paper describes the application of nanobodies as a new class of recombinant antibodies in COVID-19 treatment.

Recombinant nanobody against MUC1 tandem repeats inhibits growth, invasion, metastasis, and vascularization of spontaneous mouse mammary tumors.

Alteration in glycosylation pattern of MUC1 mucin tandem repeats during carcinomas has been shown to negatively affect adhesive properties of malignant cells and enhance tumor invasiveness and metastasis. In addition, MUC1 overexpression is closely interrelated with angiogenesis, making it a great target for immunotherapy. Alongside, easier interaction of nanobodies (single-domain antibodies) with their antigens, compared to conventional antibodies, is usually associated with superior desirable results. Herein, we evaluated the preclinical efficacy of a recombinant nanobody against MUC1 tandem repeats in suppressing tumor growth, angiogenesis, invasion, and metastasis. Expressed nanobody demonstrated specificity only toward MUC1-overexpressing cancer cells and could internalize in cancer cell lines. The IC50 values (the concentration at which the nanobody exerted half of its maximal inhibitory effect) of the anti-MUC1 nanobody against MUC1-positive human cancer cell lines ranged from 1.2 to 14.3 nm. Similar concentrations could also effectively induce apoptosis in MUC1-positive cancer cells but not in normal cells or MUC1-negative human cancer cells. Immunohistochemical staining of spontaneously developed mouse breast tumors prior to in vivo studies confirmed cross-reactivity of nanobody with mouse MUC1 despite large structural dissimilarities between mouse and human MUC1 tandem repeats. In vivo, a dose of 3 µg nanobody per gram of body weight in tumor-bearing mice could attenuate tumor progression and suppress excessive circulating levels of IL-1a, IL-2, IL-10, IL-12, and IL-17A pro-inflammatory cytokines. Also, a significant decline in expression of Ki-67, MMP9, and VEGFR2 biomarkers, as well as vasculogenesis, was evident in immunohistochemically stained tumor sections of anti-MUC1 nanobody-treated mice. In conclusion, the anti-MUC1 tandem repeat nanobody of the present study could effectively overcome tumor growth, invasion, and metastasis.