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The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the major target for antibody therapeutics. Shark-derived variable domains of new antigen receptors (VNARs) are the smallest antibody fragments with flexible paratopes that can recognize protein motifs inaccessible to classical antibodies. This study reported four VNARs binders (JM-2, JM-5, JM-17, and JM-18) isolated from Chiloscyllium plagiosum immunized with SARS-CoV-2 RBD. Biolayer interferometry showed that the VNARs bound to the RBD with an affinity KD ranging from 38.5 to 2720 nM, and their Fc fusions had over ten times improved affinity. Gel filtration chromatography revealed that JM-2-Fc, JM-5-Fc, and JM-18-Fc could form stable complexes with RBD in solution. In addition, five bi-paratopic VNARs, named JM-2-5, JM-2-17, JM-2-18, JM-5-18, and JM-17-18, were constructed by fusing two VNARs targeting distinct RBD epitopes based on epitope grouping results. All these bi-paratopic VNARs except for JM-5-18 showed higher RBD binding affinities than its component VNARs, and their Fc fusions exhibited further enhanced binding affinities, with JM-2-5-Fc, JM-2-17-Fc, JM-2-18-Fc, and JM-5-18-Fc having KD values lower than 1 pM. Among these Fc fusions of bi-paratopic VNARs, JM-2-5-Fc, JM-2-17-Fc, and JM-2-18-Fc could block the angiotensin-converting enzyme 2 (ACE2) binding to the RBD of SARS-CoV-2 wildtype, Delta, Omicron, and SARS-CoV, with inhibition rates of 48.9~84.3%. Therefore, these high-affinity VNAR binders showed promise as detectors and therapeutics of COVID-19.

The H9N2 subtype avian influenza virus, as a mutant strain of low-pathogenic avian influenza (LPAI), has augmented its transmission ability and pathogenicity via genetic evolution. At present, it has incurred the most substantial economic losses to the global poultry industry, particularly in Asia where it is extensively prevalent. To tackle this challenge, it is essential to devise effective prevention and control strategies for the H9N2 subtype. Among these strategies, vaccine development and highly sensitive diagnostic techniques are the primary countermeasures. Although more than twenty different vaccines and diagnostic kits for various types of avian influenza are currently available on the market, existing detection methods lack sufficient specificity for avian influenza virus subtypes. Precise subtype differentiation still depends on polymerase chain reaction (PCR)-based methods, which necessitate specialized equipment and are rarely accessible in on-site settings such as poultry farms. Our laboratory has developed specific single-domain antibodies (VNAR) derived from sharks against the H9N2 subtype of the avian influenza virus. The recombinant anti-H9N2 VNAR demonstrates high specificity for hemagglutinin (HA) binding activity and can specifically recognize, bind to, and neutralize the H9N2 subtype avian influenza virus on the surface of Madin-Darby canine kidney (MDCK) cells. Owing to their low molecular weight, excellent stability, and enhanced tissue penetration, these specific VNAR molecules possess extremely high application potential, rendering them innovative candidate drugs for the diagnosis and prevention of H9N2 infections.

Single-domain antibodies derive from the heavy-chain-only antibodies of Camelidae (camel, dromedary, llama, alpaca, vicuñas, and guananos; i.e., nanobodies) and cartilaginous fishes (i.e., VNARs). Their small size, antigen specificity, plasticity, and potential to recognize unique conformational epitopes represent a diagnostic and therapeutic opportunity for many central nervous system (CNS) pathologies. However, the blood–brain barrier (BBB) poses a challenge for their delivery into the brain parenchyma. Nevertheless, numerous neurological diseases and brain pathologies, including cancer, result in BBB leakiness favoring single-domain antibodies uptake into the CNS. Some single-domain antibodies have been reported to naturally cross the BBB. In addition, different strategies and methods to deliver both nanobodies and VNARs into the brain parenchyma can be exploited when the BBB is intact. These include device-based and physicochemical disruption of the BBB, receptor and adsorptive-mediated transcytosis, somatic gene transfer, and the use of carriers/shuttles such as cell-penetrating peptides, liposomes, extracellular vesicles, and nanoparticles. Approaches based on single-domain antibodies are reaching the clinic for other diseases. Several tailoring methods can be followed to favor the transport of nanobodies and VNARs to the CNS, avoiding the limitations imposed by the BBB to fulfill their therapeutic, diagnostic, and theragnostic promises for the benefit of patients suffering from CNS pathologies.

Elasmobranchs are crucial for comparative studies of evolution, as they belong to the most ancient vertebrate lineages that survived numerous extinction events and persist until today. The immunoglobulin new antigen receptor (IgNAR) found in sharks and heavy-chain-only antibody (HCAb) found in camelidae are products of convergent evolution. Although it was previously believed that IgNAR emerged 220 million years ago, before the divergence of sharks and skates, there is limited evidence to support this. In this study, we provide data supporting the existence of IgNAR in the ocellate spot skate ( Okamejei kenojei ) mononuclear cell transcriptome and peripheral blood serum. Additionally, we characterize the germline gene configuration of the ocellate spot skate IgNAR V domain. The ocellate spot skate IgNAR structure prediction and VNAR crystal structure exhibit high similarity to their shark counterparts. These data strongly suggest that IgNAR in both sharks and skates share a common ancestor. Sequencing of the ocellate spot skate VNAR repertoire provided crucial data for further understanding of the IgNAR generation. Notably, we discovered that approximately 99% of the ocellate spot skate VNARs belonged to type IV. This represents an exceptionally high proportion of type IV within the VNAR repertoire, which has not been documented in previously studied elasmobranchs. This unique characteristic of the ocellate spot skate VNAR adds essential structural diversity to the naïve VNAR library from elasmobranchs and could potentially benefit the development of pharmaceutical drugs.

Passive immunotherapy, i.e., treatment with therapeutic antibodies, has been increasingly used over the last decade in several diseases such as cancers or inflammation. However, these proteins have some limitations that single-domain antibodies could potentially solve. One of the main issues of conventional antibodies is their limited brain penetration because of the blood–brain barrier (BBB). In this review, we aim at exploring the different options single-domain antibodies (sDAbs) such as variable domain of heavy-chain antibodies (VHHs) and variable new antigen receptors (VNARs) have already taken to reach the brain allowing them to be used as therapeutic, diagnosis or transporter tools.

Single domain shark antibodies that bind to the transferrin receptor 1 (TfR1) on brain endothelial cells have been used to shuttle antibodies and other cargos across the blood brain barrier (BBB) to the brain. For these studies the TXB4 brain shuttle was fused to a TrkB neurotrophin receptor agonist antibody. The TXB4-TrkB fusion retained potent agonist activity at its cognate receptor and after systemic administration showed a 12-fold increase in brain levels over the unmodified antibody. Only the TXB4-TrkB antibody fusion was detected within the brain and localized to TrkB positive cells in the cortex and tyrosine hydroxylase (TH) positive dopaminergic neurons in the substantia nigra pars compacta (SNc), where it was associated with activated ERK1/2 signaling. When tested in the 6-hydroxydopamine (6-OHDA) mouse model of Parkinson’s disease (PD), TXB4-TrkB, but not the unmodified antibody, completely prevented the 6-OHDA induced death of TH positive neurons in the SNc. In conclusion, the fusion of the TXB4 brain shuttle allows a TrkB agonist antibody to reach neuroprotective concentrations in the brain parenchyma following systemic administration.

The variable domain of IgNAR shows great potential in biological medicine and therapy. IgNAR has been discovered in sharks and rays, with the nurse shark (Ginglymostoma cirratum) IgNARs being the most extensively studied among sharks. Despite being identified in nurse sharks over 30 years ago, the characteristics and genomic localization of IgNAR remain poorly defined, with significant gaps even in the latest released genome data. In our research, we localized the IgNAR loci in the nurse shark genome and resolved the previously missing regions. We identified three IgNAR loci, designated GcIgNAR1, GcIgNAR2, and GcIgNAR3, with only GcIgNAR1 and GcIgNAR2 being expressed. GcIgNAR1 and GcIgNAR2 belong to type I and type II IgNARs, respectively, and each exhibits several different isoforms. Most nurse shark IgNARs possess five constant domains. However, we found transcripts of GcIgNAR1 and GcIgNAR2 lacking two constant domains, C4 and C5, which differ from the IgNAR of the whitespotted bamboo shark. The protein structures of GcIgNAR1 and GcIgNAR2, generated by AlphaFold3, confirmed the accuracy of the IgNAR loci we identified. Our findings advance scientific understanding of IgNAR in nurse sharks and facilitate future research and medical applications.

Sharks and other cartilaginous fish produce new antigen receptor (IgNAR) antibodies, as key part of their humoral immune response and are the phylogenetically oldest living organisms that possess an immunoglobulin (Ig)-based adaptive immune system. IgNAR antibodies are naturally occurring heavy-chain-only antibodies, that recognize antigens with their single domain variable regions (VNARs). In this study, we structurally and biophysically elucidate the effect of antibody humanization of a previously published spiny dogfish VNAR (parent E06), which binds with high affinity to the human serum albumin (HSA). We analyze different humanization variants together with the parental E06 VNAR and the human Vκ1 light chain germline DPK9 antibody to characterize the influence of point mutations in the framework and the antigen binding site on the specificity of VNARs as reported by Kovalenko et al. We find substantially higher flexibility in the humanized variants, reflected in a broader conformational space and a higher conformational entropy, as well as population shifts of the dominant binding site ensembles in solution. A further variant, in which some mutations are reverted, largely restores the conformational stability and the dominant binding minimum of the parent E06. We also identify differences in surface hydrophobicity between the human Vκ1 light chain germline DPK9 antibody, the parent VNAR E06 and the humanized variants. Additional simulations of VNAR-HSA complexes of the parent E06 VNAR and a humanized variant reveal that the parent VNAR features a substantially stronger network of stabilizing interactions. Thus, we conclude that a structural and dynamic understanding of the VNAR binding site upon humanization is a key aspect in antibody humanization.

Severe Acute Respiratory Syndrome Coronavirus 2 is the causal pathogen of coronavirus disease 2019 (COVID-19). The emergence of new variants with different mutational patterns has limited the therapeutic options available and complicated the development of effective neutralizing antibodies targeting the spike (S) protein. Variable New Antigen Receptors (VNARs) constitute a neutralizing antibody technology that has been introduced into the list of possible therapeutic options against SARS-CoV-2. The unique qualities of VNARs, such as high affinities for target molecules, capacity for paratope reformatting, and relatively high stability, make them attractive molecules to counteract the emerging SARS-CoV-2 variants. In this study, we characterized a VNAR antibody (SP240) that was isolated from a synthetic phage library of VNAR domains. In the phage display, a plasma with high antibody titers against SARS-CoV-2 was used to selectively displace the VNAR antibodies bound to the antigen SARS-CoV-2 receptor binding domain (RBD). In silico data suggested that the SP240 binding epitopes are located within the ACE2 binding interface. The neutralizing ability of SP240 was tested against live Delta and Omicron SARS-CoV-2 variants and was found to clear the infection of both variants in the lung cell line A549-ACE2-TMPRSS2. This study highlights the potential of VNARs to act as neutralizing antibodies against emerging SARS-CoV-2 variants.

Glycosaminoglycans (GAGs) are key ligands for proteins involved in physiological and pathological processes. Specific GAG-binding patterns are rarely identified, with the heparin pentasaccharide as an Antithrombin-III ligand being the best characterized. Generating glycan-specific antibodies is difficult due to their size, pattern dispersion, and flexibility. Single-domain variable new antigen receptors (VNAR nanobodies) from nurse sharks are highly soluble, stable, and versatile. Their unique properties suggest advantages over conventional antibodies, particularly for challenging biotherapeutic targets. Here we have used VNAR semi-synthetic phage libraries to select high-affinity fondaparinux-binding VNARs that did not show cross-reactivity with other GAG species. Competition ELISA and surface plasmon resonance identified a single fondaparinux-selective VNAR clone. This VNAR exhibited an extraordinarily stable protein fold: the beta-strands are stabilized by a robust hydrophobic network, as revealed by heteronuclear NMR. Docking fondaparinux to the VNAR structure revealed a large contact surface area between the CDR3 loop of the antibody and the glycan. Fusing the VNAR with a human Fc domain resulted in a stable product with a high affinity for fondaparinux (Kd = 9.3 × 10−8 M) that could efficiently discriminate between fondaparinux and other glycosaminoglycans. This novel glycan-targeting screening technology represents a promising therapeutic strategy for addressing GAG-related diseases.