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In response to the invasion of exogenous microorganisms, one of the defence strategies of the immune system is to produce antibodies. Cartilaginous fish is among those who evolved the earliest humoral immune system that utilizes immunoglobulin-type antibodies. The cartilaginous fish antibodies fall into three categories: IgW, IgM, and IgNAR. The shark Immunoglobulin Novel Antigen Receptor (IgNAR) constitutes disulfide-bonded dimers of two protein chains, similar to the heavy chain of mammalian IgGs. Shark IgNAR is the primary antibody of a shark's adaptive immune system with a serum concentration of 0.1–1.0 mg/mL. Its structure comprises of one variable (V) domain (VNAR) and five constant (C1 –C5) domains in the secretory form. VNARs are classified into several subclasses based on specific properties such as the quantity and position of additional non-canonical cysteine (Cys) residues in the VNAR. The VDJ recombination in IgNAR comprises various fragments; one variable component, three diverse sections, one joining portion, and a solitary arrangement of constant fragments framed in each IgNAR gene cluster. The re-arrangement happens just inside this gene cluster bringing about a VD1D2D3J segment. Therefore, four re-arrangement procedures create the entire VNAR space. IgNAR antibody can serve as an excellent diagnostic, therapeutic, and research tool because it has a smaller size, high specificity for antigen-binding, and perfect stability. The domain characterization, structural features, types, diversity and therapeutic applications of IgNAR molecules are highlighted in this review. It would be helpful for further research on IgNAR antibodies acting as an essential constituent of the adaptive immune system and a potential therapeutic agent. • Cartilaginous fish antibodies, namely IgW, IgM, IgNAR, are reviewed. • The molecular properties of VNARs, including domain organization, structural features are summarized. • VNAR shuttles are excellent candidates for therapeutic development against neurodegenerative diseases. • New therapeutic approaches have been aimed to design SARS-COV-2 neutralizing VNAR antibodies. [ABSTRACT FROM AUTHOR]

IgNAR exhibits significant promise in the fields of cancer and anti-virus biotherapies. Notably, the variable regions of IgNAR (VNAR) possess comparable antigen binding affinity with much smaller molecular weight (∼12 kDa) compared to IgNAR. Antigen specific VNAR screening is a changeling work, which limits its application in medicine and therapy fields. Though phage display is a powerful tool for VNAR screening, it has a lot of drawbacks, such as small library coverage, low expression levels, unstable target protein, complicating and time-consuming procedures. Here we report VANR screening with next generation sequencing (NGS) could effectively overcome the limitations of phage display, and we successfully identified approximately 3000 BAFF-specific VNARs in Chiloscyllium plagiosum vaccinated with the BAFF antigen. The results of modelling and molecular dynamics simulation and ELISA assay demonstrated that one out of the top five abundant specific VNARs exhibited higher binding affinity to the BAFF antigen than those obtained through phage display screening. Our data indicates NGS would be an alternative way for VNAR screening with plenty of advantages. • Next Generation Sequencing (NGS) was successfully employed as an alternative method for screening antigen-specific VNARs. • Through NGS screening, approximately 3000 BAFF-specific VNARs were identified in Whitespotted Bamboo Shark. • Simulation techniques were utilized to evaluate the binding affinity of VNARs to the antigen. [ABSTRACT FROM AUTHOR]

Single domain antibodies (sdAb) are promising candidates in cancer and anti-virus biotherapies for their unique structure characters. Though VHH and IgNAR have been discovered in camelidae and nurse shark (Ginlymostoma cirratum) respectively serval decades ago, expense of these large animals still limits the studies and applications of sdAb. Recently, IgNAR has been found in whitespotted bamboo shark (Chiloscyllium plagiosum), a small-sized sharks, while how to characterize and achieved the IgNAR of whitespotted bamboo shark is still unclear. In our research, we identified four IgNAR coding gene loci in whitespotted bamboo shark chromosome 44 (NC_057753.1), and primers were designed for single domain variable regions of IgNAR (VNAR) libraries preparation. Following sequencing results revealed that all plasmids constructed with our predicted VNAR libraries contained VNAR coding sequences, which confirmed the specificities of our primers in VNAR amplification. To our surprise, ≥90% VNAR sequences were encoded by IgNAR1 , which suggests IgNAR1 is the most active IgNAR transcription locus in whitespotted bamboo shark. Interestingly, we found IgNAR(ΔC2-C3) were encoded by IgNAR3. Our findings gave a new sight of whitespotted bamboo shark IgNAR, which would broad the way of IgNAR studies and applications in biotherapies. • Four IgNAR coding gene loci in whitespotted bamboo shark have been identified. • ≥90% VNAR sequences were encoded by IgNAR1 , makes it the most active IgNAR transcription locus. • Novel VNAR type of FR4-missing has been firstly reported. [ABSTRACT FROM AUTHOR]

Abstract: The shark antigen-binding VNAR domain has the potential to provide an attractive alternative to traditional biotherapeutics based on its small size, advantageous physiochemical properties, and unusual ability to target clefts in enzymes or cell surface molecules. The VNAR shares many of the properties of the well-characterised single-domain camelid VHH but is much less understood at the molecular level. We chose the hen-egg-lysozyme-specific archetypal Type I VNAR 5A7 and used ribosome display in combination with error-prone mutagenesis to interrogate the entire sequence space. We found a high level of mutational plasticity across the VNAR domain, particularly within the framework 2 and hypervariable region 2 regions. A number of residues important for affinity were identified, and a triple mutant combining A1D, S61R, and G62R resulted in a K D of 460 pM for hen egg lysozyme, a 20-fold improvement over wild-type 5A7, and the highest K D yet reported for VNAR–antigen interactions. These findings were rationalised using structural modelling and indicate the importance of residues outside the classical complementarity determining regions in making novel antigen contacts that modulate affinity. We also located two solvent-exposed residues (G15 and G42), distant from the VNAR paratope, which retain function upon mutation to cysteine and have the potential to be exploited as sites for targeted covalent modification. Our findings with 5A7 were extended to all known NAR structures using an in-depth bioinformatic analysis of sequence data available in the literature and a newly generated VNAR database. This study allowed us to identify, for the first time, both VNAR-specific and VNAR/Ig VL/TCR Vα overlapping hallmark residues, which are critical for the structural and functional integrity of the single domain. Intriguingly, each of our designated VNAR-specific hallmarks align precisely with previously defined mutational ‘cold spots’ in natural nurse shark cDNA sequences. These findings will aid future VNAR engineering and optimisation studies towards the development of VNAR single-domain proteins as viable biotherapeutics. [Copyright &y& Elsevier]