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Introduction: B-cell lymphoma, a malignant proliferative disease originating from lymphoid tissue, poses a grave threat to human health. CD20 has emerged as a promising target for lymphoma treatment. However, due to the significant heterogeneity of B-cell lymphomas, conventional CD20 monoclonal antibodies show limited penetration, severely impeding the progress of B-cell lymphoma therapies. Methods: In contrast, single-domain antibody molecules derived from cartilaginous fish have a molecular weight as small as 12 kDa, granting them robust penetration capabilities and making them the smallest known molecules of efficiently targeting specific antigens. Results: As a result, these molecules hold tremendous potential as candidate drugs for lymphoma treatment. In this study, the whitespotted bamboo shark (Chiloscyllium plagiosum) was immunized with recombinant human CD20 to generate specific single-domain antibodies (sdAbs) targeting CD20. By utilizing phage display technology, the variable new antigen receptors (VNARs) were successfully screened and identified, and play an important role in the inhibition of Raji lymphoblastoma. Discussion: The sdAbs obtained through this research represent promising candidates for B-cell lymphoma treatment, displaying significant potential for clinical applications and offering a new direction for the development of targeted therapies against lymphoma. [ABSTRACT FROM AUTHOR]

The editorial in Frontiers in Immunology discusses the evolution of single-domain antibodies (sdAbs) since 2017, highlighting their significant impact in various fields, including drug development, cell therapy, and imaging. The editorial emphasizes the distinct properties of sdAbs compared to conventional antibodies and their potential in diagnostics and therapeutics. It also explores the use of sdAbs in non-invasive imaging, particularly for detecting SARS-CoV-2 and other pathogens. The editorial concludes by acknowledging the regulatory approvals of sdAb-based drugs and the ongoing efforts to further understand and utilize these unique molecules effectively. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

Single domain antibody (sdAb) is only composed of a variable domain of the heavy-chain-only antibody, which is devoid of light chain and naturally occurring in camelids and cartilaginous fishes. Variable New Antigen Receptor (VNAR), a type of single domain antibody present in cartilaginous fishes such as sharks, is the smallest functional antigen-binding fragment found in nature. The unique features, including flexible paratope, high solubility and outstanding stability make VNAR a promising prospect in antibody drug development and structural biology research. However, VNAR's research has lagged behind camelid-derived sdAb, especially in the field of structural research. Here we report the 1H,15N,13C resonance assignments of a VNAR derived from the immune library of Chiloscyllium plagiosum, termed B2-3, which recognizes the hyaluronan synthase. Analysis of the backbone chemical shifts demonstrates that the secondary structure of VNAR is predominately composed of β-sheets corresponding to around 40% of the B2-3 backbone. The Cβ chemical shift values of cysteine residues, combined with mass spectrometry data, clearly shows that B2-3 contains two pairs of disulfide bonds, which is import for protein stability. The assignments will be essential for determining the high resolution solution structure of B2-3 by NMR spectroscopy. [ABSTRACT FROM AUTHOR]

IntroductionB-cell lymphoma, a malignant proliferative disease originating from lymphoid tissue, poses a grave threat to human health. CD20 has emerged as a promising target for lymphoma treatment. However, due to the significant heterogeneity of B-cell lymphomas, conventional CD20 monoclonal antibodies show limited penetration, severely impeding the progress of B-cell lymphoma therapies.MethodsIn contrast, single-domain antibody molecules derived from cartilaginous fish have a molecular weight as small as 12 kDa, granting them robust penetration capabilities and making them the smallest known molecules of efficiently targeting specific antigens.ResultsAs a result, these molecules hold tremendous potential as candidate drugs for lymphoma treatment. In this study, the whitespotted bamboo shark (Chiloscyllium plagiosum) was immunized with recombinant human CD20 to generate specific single-domain antibodies (sdAbs) targeting CD20. By utilizing phage display technology, the variable new antigen receptors (VNARs) were successfully screened and identified, and play an important role in the inhibition of Raji lymphoblastoma.DiscussionThe sdAbs obtained through this research represent promising candidates for B-cell lymphoma treatment, displaying significant potential for clinical applications and offering a new direction for the development of targeted therapies against lymphoma.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously “undruggable” targets for biologic-based therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously “undruggable” targets for biologic-based therapy.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generated a joint global effort to develop vaccines and other treatments that could mitigate the negative effects and the rapid spread of the virus. Single-domain antibodies derived from various sources, including cartilaginous fish, camelids, and humans, have gained attention as promising therapeutic tools against coronavirus disease 2019. Shark-derived variable new antigen receptors (VNARs) have emerged as the smallest naturally occurring antigen-binding molecules. Here, we compile and review recent published studies on VNARs with the capacity to recognize and/or neutralize SARS-CoV-2. We found a close balance between the use of natural immune libraries and synthetic VNAR libraries for the screening against SARS-CoV-2, with phage display being the preferred display technology for the selection of VNARs against this virus. In addition, we discuss potential modifications and engineering strategies employed to improve the neutralization potential of VNARs, such as exploring fusion with the Fc domain of human Immunoglobulin G (IgG) to increase avidity and therapeutic potential. This research highlights the potential of VNARs as powerful molecular tools in the fight against infectious diseases. [ABSTRACT FROM AUTHOR]

Glioblastoma is a common and fatal malignant tumour of the central nervous system, with high invasiveness. Conventional treatments for this disease, including comprehensive treatment of surgical resection combined with chemoradiotherapy, are ineffective, with low survival rate and extremely poor prognosis. Targeted therapy is promising in overcoming the difficulties in brain tumour treatment and IL‐13Rα2 is a widely watched target. The development of new therapies for glioma, however, is challenged by factors, such as the unique location and immune microenvironment of gliomas. The unique advantages of single‐domain antibodies (sdAbs) may provide a novel potential treatment for brain tumours. In this study, Chiloscyllium plagiosum was immunized with recombinant IL‐13Rα2 protein to produce sdAb and sdAb sequences were screened by multi‐omics. The targeted sdAb genes obtained were efficiently expressed in the Escherichia coli prokaryotic expression system, showing a significant binding capacity to IL‐13Rα2 in vitro. The cell proliferation and migration inhibitory effects of recombinant variable domain of the new antigen receptor (VNAR) on glioma cells were detected by CCK‐8 and cell scratch assays. The sdAb obtained in this study showed high in vitro activity and favourable cell proliferation inhibitory effect on glioma cells, with potential clinical application value. The present study also provides a new direction and experimental basis for the development of targeted therapies for glioma. [ABSTRACT FROM AUTHOR]

Antibodies represent a relatively mature detection means and serve as therapeutic drug carriers in the clinical diagnosis and treatment of cancer—among which monoclonal antibodies (mAbs) currently occupy a dominant position. However, the emergence and development of small-molecule monodomain antibodies are inevitable due to the many limitations of mAbs, such as their large size, complex structure, and sensitivity to extreme temperature, and tumor microenvironments. Thus, since first discovered in Chondroid fish in 1995, IgNAR has become an alternative therapeutic strategy through which to replace monoclonal antibodies, thus entailing that this novel type of immunoglobulin has received wide attention with respect to clinical diagnoses and tumor therapies. The variable new antigen receptor (VNAR) of IgNAR provides an advantage for the development of new antitumor drugs due to its small size, high stability, high affinity, as well as other structural and functional characteristics. In that respect, a better understanding of the unique characteristics and therapeutic potential of IgNAR/VNAR in clinical and anti-tumor treatment is needed. This article reviews the advantages of its unique biochemical conditions and molecular structure for clinical diagnoses and novel anti-tumor drugs. At the same time, the main advantages of the existing conjugated drugs, which are based on single-domain antibodies, are introduced here, thereby providing new ideas and methods for the development of clinical diagnoses and anti-tumor therapies in the future. [ABSTRACT FROM AUTHOR]

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generated a joint global effort to develop vaccines and other treatments that could mitigate the negative effects and the rapid spread of the virus. Single-domain antibodies derived from various sources, including cartilaginous fish, camelids, and humans, have gained attention as promising therapeutic tools against coronavirus disease 2019. Shark-derived variable new antigen receptors (VNARs) have emerged as the smallest naturally occurring antigen-binding molecules. Here, we compile and review recent published studies on VNARs with the capacity to recognize and/or neutralize SARS-CoV-2. We found a close balance between the use of natural immune libraries and synthetic VNAR libraries for the screening against SARS-CoV-2, with phage display being the preferred display technology for the selection of VNARs against this virus. In addition, we discuss potential modifications and engineering strategies employed to improve the neutralization potential of VNARs, such as exploring fusion with the Fc domain of human Immunoglobulin G (IgG) to increase avidity and therapeutic potential. This research highlights the potential of VNARs as powerful molecular tools in the fight against infectious diseases.

The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10−8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning.

Immunoglobulin new antigen receptor (IgNAR) is a naturally occurring antibody that consists of only two heavy chains with two independent variable domains. The variable binding domain of IgNAR, called variable new antigen receptor (VNAR), is attractive due to its solubility, thermal stability, and small size. Hepatitis B surface antigen (HBsAg) is a viral capsid protein found on the surface of the Hepatitis B virus (HBV). It appears in the blood of an individual infected with HBV and is widely used as a diagnostic marker for HBV infection. In this study, the whitespotted bamboo sharks (Chiloscyllium plagiosum) were immunized with the recombinant HBsAg protein. Peripheral blood leukocytes (PBLs) of immunized bamboo sharks were further isolated and used to construct a VNAR-targeted HBsAg phage display library. The 20 specific VNARs against HBsAg were then isolated by bio-panning and phage ELISA. The 50% of maximal effect (EC50) of three nanobodies, including HB14, HB17, and HB18, were 4.864 nM, 4.260 nM, and 8.979 nM, respectively. The Sandwich ELISA assay further showed that these three nanobodies interacted with different epitopes of HBsAg protein. When taken together, our results provide a new possibility for the application of VNAR in HBV diagnosis and also demonstrate the feasibility of using VNAR for medical testing. [ABSTRACT FROM AUTHOR]

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.

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.

Antibodies represent a relatively mature detection means and serve as therapeutic drug carriers in the clinical diagnosis and treatment of cancer—among which monoclonal antibodies (mAbs) currently occupy a dominant position. However, the emergence and development of small-molecule monodomain antibodies are inevitable due to the many limitations of mAbs, such as their large size, complex structure, and sensitivity to extreme temperature, and tumor microenvironments. Thus, since first discovered in Chondroid fish in 1995, IgNAR has become an alternative therapeutic strategy through which to replace monoclonal antibodies, thus entailing that this novel type of immunoglobulin has received wide attention with respect to clinical diagnoses and tumor therapies. The variable new antigen receptor (VNAR) of IgNAR provides an advantage for the development of new antitumor drugs due to its small size, high stability, high affinity, as well as other structural and functional characteristics. In that respect, a better understanding of the unique characteristics and therapeutic potential of IgNAR/VNAR in clinical and anti-tumor treatment is needed. This article reviews the advantages of its unique biochemical conditions and molecular structure for clinical diagnoses and novel anti-tumor drugs. At the same time, the main advantages of the existing conjugated drugs, which are based on single-domain antibodies, are introduced here, thereby providing new ideas and methods for the development of clinical diagnoses and anti-tumor therapies in the future.

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. [ABSTRACT FROM AUTHOR]

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 Vk1 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. [ABSTRACT FROM AUTHOR]

Single‐domain antibodies (sdAbs), the autonomous variable domains of camelid and shark heavy‐chain antibodies, have many desirable properties as components of biologic drugs. However, their sequences may increase the risk of immunogenicity and antidrug antibody (ADA) development in humans, and thus, sdAbs are routinely humanized during development. Here, we review and summarize the available evidence regarding the factors governing immunogenicity of sdAbs and our current state of knowledge of strategies to mitigate immunogenicity risks by humanization. While several sdAb properties, including high homology of camelid VHHs with human IGHV3 gene products, favor low immunogenicity in humans, epitopes absent in the human repertoire including the exposed VH:VL interface may be intrinsically immunogenic. While most clinical trials have demonstrated minimal sdAb immunogenicity, two notable exceptions (the tetrameric DR5‐specific VHH TAS266 and the TNFR1‐specific VH GSK1995057) illustrate that special caution must be taken in identifying preexisting ADAs against highly potent sdAbs. Nonhuman sequence alone does not adequately explain sdAb immunogenicity, as some camelid VHHs are nonimmunogenic while some fully human VHs elicit ADAs. The presence of preexisting ADAs directed against the exposed C‐termini of some sdAbs in a significant proportion of individuals awaits a molecular explanation. Whether sdAb humanization reduces or promotes immunogenicity remains unclear: reduction of nonhuman sequence content at the expense of introducing low‐level aggregation in humanized variants may be counterproductive. Further work will establish thresholds for VHH and VNAR humanization to maximize human sequence content while avoiding loss of binding affinity and/or immunogenicity resulting from aggregation or decreased stability. [ABSTRACT FROM AUTHOR]

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.

Tumor necrosis factor α (TNFα), an important clinical testing factor and drug target, can trigger serious autoimmune diseases and inflammation. Thus, the TNFα antibodies have great potential application in diagnostics and therapy fields. The variable binding domain of IgNAR (VNAR), the shark single domain antibody, has some excellent advantages in terms of size, solubility, and thermal and chemical stability, making them an ideal alternative to conventional antibodies. This study aims to obtain VNARs that are specific for mouse TNF (mTNF) from whitespotted bamboosharks. After immunization of whitespotted bamboosharks, the peripheral blood leukocytes (PBLs) were isolated from the sharks, then the VNAR phage display library was constructed. Through phage display panning against mTNFα, positive clones were validated through ELISA assay. The affinity of the VNAR and mTNFα was measured using ELISA and Bio-Layer Interferometry. The binding affinity of 3B11 VNAR reached 16.7 nM. Interestingly, one new type of VNAR targeting mTNF was identified that does not belong to any known VNAR type. To understand the binding mechanism of VNARs to mTNFα, the models of VNARs-mTNFα complexes were predicted by computational modeling combining HawkDock and RosettaDock. Our results showed that four VNARs' epitopes overlapped in part with that of mTNFR. Furthermore, the ELISA assay shows that the 3B11 potently inhibited mTNFα binding to mTNFR. This study may provide the basis for the TNFα blockers and diagnostics applications. [ABSTRACT FROM AUTHOR]

Immunoglobulin new antigen receptor (IgNAR) is a naturally occurring antibody that consists of only two heavy chains with two independent variable domains. The variable binding domain of IgNAR, called variable new antigen receptor (VNAR), is attractive due to its solubility, thermal stability, and small size. Hepatitis B surface antigen (HBsAg) is a viral capsid protein found on the surface of the Hepatitis B virus (HBV). It appears in the blood of an individual infected with HBV and is widely used as a diagnostic marker for HBV infection. In this study, the whitespotted bamboo sharks (Chiloscyllium plagiosum) were immunized with the recombinant HBsAg protein. Peripheral blood leukocytes (PBLs) of immunized bamboo sharks were further isolated and used to construct a VNAR-targeted HBsAg phage display library. The 20 specific VNARs against HBsAg were then isolated by bio-panning and phage ELISA. The 50% of maximal effect (EC50) of three nanobodies, including HB14, HB17, and HB18, were 4.864 nM, 4.260 nM, and 8.979 nM, respectively. The Sandwich ELISA assay further showed that these three nanobodies interacted with different epitopes of HBsAg protein. When taken together, our results provide a new possibility for the application of VNAR in HBV diagnosis and also demonstrate the feasibility of using VNAR for medical testing.

Recently, the incidence of malignant tumors is on the rise and searching for new treatments on it has become the research priority. Blocking the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is one of the treatment strategies that used in the development of specific anti-angiogenic drugs. The deficiencies in tissue penetration and affinity maturation become the weakness of these drugs in anti-tumors applications. The single heavy chain antibody found in Chiloscyllium plagiosum, which has a low molecular weight and superior tissue penetration of variable region (variable new antigen receptor, VNARs), was considered to have the high antigen-binding activity and stability. This type of antibody has a simple structure that can be prokaryoticaly expressed, which makes it easily to produce new antiangiogenic target drugs. Specific anti-IgNAR rabbit multiple antibodies have been used to assess the level of VNARs in sharks and have shown a significant enrichment of IgNAR after triple immunization. An anti-VEGFR2 phage library was used for the targeted VNARs screening, and five candidate VNARs sequences were subsequently obtained by phage screening, followed by combined screening with the transcriptome library, and analysis of conserved regions along with 3D modelling matched the VNAR profile. ELISA and cell-based assays showed that two of the VNARs, VNAR-A6, and VNAR-E3, had a superior antigen affinity and anti-angiogenic activity thereby being able to inhibit human Umbilical Vein Endothelial Cells proliferation and migration. The anti-VEGFR2 VNARs derived from the immunized C. plagiosum and screened by phage library, which provide the new research ideas and specific approaches for the development of new drugs. The anti-VEGFR2 VNARs are capable for blocking the VEGF-VEGFR pathway, which of these may contribute to expanding the use of anti-angiogenic drugs., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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]

The development of shark single domain antibodies (sdAbs) is hindered by the high cost and tediousness of large-sized shark farming. Here, we demonstrated white-spotted bamboo sharks (Chiloscyllium plagiosum) being cultivated commercially as a promising small animal model to produce sdAbs. We found that immunoglobulin new antigen receptor (IgNAR) presented in bamboo shark genome, transcriptome, and plasma. Four complete IgNAR clusters including variable domains (vNARs) were discovered in the germline, and the Variable–Joining pair from IgNAR1 cluster was dominant from immune repertoires in blood. Bamboo sharks developed effective immune responses upon green fluorescent protein (GFP), near-infrared fluorescent protein iRFP713, and Freund’s adjuvant immunization revealed by elevated lymphocyte counts and antigen specific IgNAR. Before and after immunization, the complementarity determining region 3 (CDR3) of IgNAR were the major determinant of IgNAR diversity revealed by 400-bp deep sequencing. To prove that bamboo sharks could produce high-affinity IgNAR, we isolated anti-GFP and anti-iRFP713 vNARs with up to 0.3 and 3.8 nM affinities, respectively, from immunized sharks. Moreover, we constructed biparatopic vNARs with the highest known affinities (20.7 pM) to GFP and validated the functions of anti-GFP vNARs as intrabodies in mammalian cells. Taken together, our study will accelerate the discovery and development of bamboo shark sdAbs for biomedical industry at low cost and easy operation.

Single domain shark variable domain of new antigen receptor (VNAR) antibodies can offer a viable alternative to conventional Ig‐based monoclonal antibodies in treating COVID‐19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the severe acute respiratory syndrome coronavirus 2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant angiotensin‐converting enzyme 2 (ACE2) receptor in vitro. In addition, three antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies, due to their low complexity, small size, unique epitope recognition, and formatting flexibility, should be a useful adjunct to existing antibody approaches to treat COVID‐19. [ABSTRACT FROM AUTHOR]

The Shark-derived immunoglobulin new antigen receptors (IgNARs) have gained increasing attention for their high solubility, exceptional thermal stability, and intricate sequence variation. In this study, we immunized whitespotted bamboo shark (Chiloscyllium plagiosum) to create phage display library of variable domains of IgNAR (VNARs) for screening against Human Serum Albumin (HSA), a versatile vehicle in circulation due to its long in vivo half-life. We identified two HSA-binding VNAR clones, 2G5 and 2G6, and enhanced their expression in E. coli with the FKPA chaperone. 2G6 exhibited a strong binding affinity of 13 nM with HSA and an EC 50 of 1 nM. In vivo study with a murine model further provided initial validation of 2G6's ability to prolong circulation time by binding to HSA. Additionally, we employed computational molecular docking to predict the binding affinities of both 2G6 and its humanized derivative, H2G6, to HSA. Our analysis unveiled that the complementarity-determining regions (CDR1 and CDR3) are pivotal in the antigen recognition process. Therefore, our study has advanced the understanding of the potential applications of VNARs in biomedical research aimed at extending drug half-life, holding promise for future therapeutic and diagnostic progressions. [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]

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that have antibodies as part of their adaptive immune system. As part of their humoral adaptive immune response, they produce an immunoglobulin, the so-called immunoglobulin new antigen receptor (IgNAR), a heavy-chain only antibody. The variable domain of an IgNAR, also known as VNAR, binds the antigen as an independent soluble domain. In this study, we structurally and dynamically characterized the affinity maturation mechanism of the germline and somatically matured (PBLA8) VNAR to better understand their function and their applicability as therapeutics. We observed a substantial rigidification upon affinity maturation, which is accompanied by a higher number of contacts, thereby contributing to the decrease in flexibility. Considering the static x-ray structures, the observed rigidification is not obvious, as especially the mutated residues undergo conformational changes during the simulation, resulting in an even stronger network of stabilizing interactions. Additionally, the simulations of the VNAR in complex with the hen egg-white lysozyme show that the VNAR antibodies evidently follow the concept of conformational selection, as the binding-competent state already preexisted even without the presence of the antigen. To have a more detailed description of antibody–antigen recognition, we also present here the binding/unbinding mechanism between the hen egg-white lysozyme and both the germline and matured VNARs. Upon maturation, we observed a substantial increase in the resulting dissociation-free energy barrier. Furthermore, we were able to kinetically and thermodynamically describe the binding process and did not only identify a two-step binding mechanism, but we also found a strong population shift upon affinity maturation toward the native binding pose.

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy.

Tumor necrosis factor α (TNFα), an important clinical testing factor and drug target, can trigger serious autoimmune diseases and inflammation. Thus, the TNFα antibodies have great potential application in diagnostics and therapy fields. The variable binding domain of IgNAR (VNAR), the shark single domain antibody, has some excellent advantages in terms of size, solubility, and thermal and chemical stability, making them an ideal alternative to conventional antibodies. This study aims to obtain VNARs that are specific for mouse TNF (mTNF) from whitespotted bamboosharks. After immunization of whitespotted bamboosharks, the peripheral blood leukocytes (PBLs) were isolated from the sharks, then the VNAR phage display library was constructed. Through phage display panning against mTNFα, positive clones were validated through ELISA assay. The affinity of the VNAR and mTNFα was measured using ELISA and Bio-Layer Interferometry. The binding affinity of 3B11 VNAR reached 16.7 nM. Interestingly, one new type of VNAR targeting mTNF was identified that does not belong to any known VNAR type. To understand the binding mechanism of VNARs to mTNFα, the models of VNARs-mTNFα complexes were predicted by computational modeling combining HawkDock and RosettaDock. Our results showed that four VNARs’ epitopes overlapped in part with that of mTNFR. Furthermore, the ELISA assay shows that the 3B11 potently inhibited mTNFα binding to mTNFR. This study may provide the basis for the TNFα blockers and diagnostics applications.

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy. [ABSTRACT FROM AUTHOR]

IgG antibodies are some of the most important biopharmaceutical molecules with a high market volume. In spite of the fact that clinical therapies with antibodies are broadly utilized in oncology, immunology and hematology, their delivery strategies and biodistribution need improvement, their limitations being due to their size and poor ability to penetrate into tissues. In view of their small size, there is a rising interest in derivatives, such as single-domain antibodies and single-chain variable fragments, for clinical diagnostic but also therapeutic applications. Smaller antibody formats combine several benefits for clinical applications and can be manufactured at reduced production costs compared with full-length IgGs. Moreover, such formats have a relevant potential for targeted drug delivery that directs drug cargo to a specific tissue or across the blood–brain barrier. In this review, we give an overview of the challenges for antibody drug delivery in general and focus on intranasal delivery to the central nervous system with antibody formats of different sizes.

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.

Phage display antibody libraries have proven an invaluable resource for the isolation of diagnostic and potentially therapeutic antibodies, the latter usually being antibody fragments converted into IgG formats. Recent advances in the production of highly diverse and functional antibody libraries are considered here, including for Fabs, scFvs and nanobodies. These advances include codon optimisation during generation of CDR diversity, improved display levels using novel signal sequences, molecular chaperones and isomerases and the use of highly stable scaffolds with relatively high expression levels. In addition, novel strategies for the batch reformatting of scFv and Fab phagemid libraries, derived from phage panning, into IgG formats are described. These strategies allow the screening of antibodies in the end-use format, facilitating more efficient selection of potential therapeutics. [ABSTRACT FROM AUTHOR]

At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest) antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

Homodimeric antibodies devoid of light chains have evolved multiple times through convergent evolution, yet their specific immunological functions remain poorly understood. We survey the molecular and structural features of these antibodies, their immunological functions in host defense, and reflect on the long-standing question of the evolutionary forces driving their emergence. [ABSTRACT FROM AUTHOR]

Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified in vivo through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length.