Screening and anti-angiogenesis activity of Chiloscyllium plagiosum anti-human VEGFR2 single-domain antibody.

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.

Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2.

SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other ß coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens.

Antigen specific VNAR screening in whitespotted bamboo shark (Chiloscyllium plagiosum) with next generation sequencing.

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.

Screening and anti-glioma activity of Chiloscyllium plagiosum anti-human IL-13Rα2 single-domain antibody.

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.

IgNAR characterization and gene loci identification in whitespotted bamboo shark (Chiloscyllium plagiosum) genome.

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.

Exploring shark VNAR antibody against infectious diseases using phage display technology.

Antibody with high affinity and specificity to antigen has widely used as a tool to combat various diseases. The variable domain of immunoglobulin new antigen receptor (VNAR) naturally found in shark contains autonomous function as single-domain antibody. Due to its excellent characteristics, the small, non-complex, and highly stable have made shark VNAR can acquires the antigen-binding capability that might not be reached by conventional antibody. Phage display technology enables shark VNAR to be presented on the surface of phage, allowing the exploration of shark VNAR as an alternative antibody format to target antigens from various infectious diseases. The application of phage-displayed shark VNAR in antibody library and biopanning eventually leads to the discovery and isolation of antigen-specific VNARs with diagnostic and therapeutic potential towards infectious diseases. This review provides an overview of the shark VNAR antibody, the types of phage display technology with comparison to the other types of display system, as well as the application and case studies of phage-displayed shark VNAR antibodies against infectious diseases.

Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein.

The COVID-19 pandemic has significantly impacted human health for three years. To mitigate the spread of SARS-CoV-2, the development of neutralizing antibodies has been accelerated, including the exploration of alternative antibody formats such as single-domain antibodies. In this study, we identified variable new antigen receptors (VNARs) specific for the receptor binding domain (RBD) of SARS-CoV-2 by immunizing a banded houndshark (Triakis scyllium) with recombinant wild-type RBD. Notably, the CoV2NAR-1 clone showed high binding affinities in the nanomolar range to various RBDs and demonstrated neutralizing activity against SARS-CoV-2 pseudoviruses. These results highlight the potential of the banded houndshark as an animal model for the development of VNAR-based therapeutics or diagnostics against future pandemics.

Isolation and Characterization of Targeting-HBsAg VNAR Single Domain Antibodies from Whitespotted Bamboo Sharks (Chiloscyllium plagiosum).

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.

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.

Shark IgNAR: The Next Broad Application Antibody in Clinical Diagnoses and Tumor Therapies?

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.

Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro.

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.

Blood-brain barrier transport using a high affinity, brain-selective VNAR antibody targeting transferrin receptor 1.

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.

IgNAR antibody: Structural features, diversity and applications.

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.

Identification of Anti-TNFα VNAR Single Domain Antibodies from Whitespotted Bambooshark (Chiloscyllium plagiosum).

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.

Screening and Characterization of Shark-Derived VNARs against SARS-CoV-2 Spike RBD Protein.

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.

Neutralizing Ability of a Single Domain VNAR Antibody: In Vitro Neutralization of SARS-CoV-2 Variants of Concern.

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.

Immunological Functions and Evolutionary Emergence of Heavy-Chain Antibodies.

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.

Cytoplasmic and periplasmic expression of recombinant shark VNAR antibody in Escherichia coli.

Shark variable new antigen receptors (VNARs) are known to possess excellent heat-stability, and the long complementarity determining region 3 (CDR3) has permitted it to penetrate into the cleft region of antigens. The number of cysteine (Cys) residues contained within VNAR is greater than in conventional antibodies, entailing disulfide bond formation in both the inter- or intra-loop regions is required for interactions with the target protein antigens. Therefore, the selection of a suitable expression system is important to ensure the solubility and correct folding of functional VNAR protein production. Unlike higher organisms, the machinery for effecting posttranslational modifications of proteins in Escherichia coli (E. coli) are less sophisticated. To overcome this circumstance, a pDSB-28Y vector fusion with DsbA signal peptide was engineered for periplasmic H8VNAR production. Despite the periplasmic proteins showing a lower yield (62 µg/mL) than cytosolic proteins (468 µg/mL) that is obtained from pET-28a vector, it has demonstrated better performance than that of a cytosolic protein in terms of absorbance. However, these readings were still inferior to that of positive control mouse monoclonal antibody (mAb) C1-13 in this experiment. Therefore, further investigation is required to improve the binding affinity of selected recombinant VNAR towards malaria biomarkers.

Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF165 Neutralization.

Variable new antigen receptor domain (vNAR) antibodies are novel, naturally occurring antibodies that can be isolated from naïve, immune or synthetic shark libraries. These molecules are very interesting to the biotechnology and pharmaceutical industries because of their unique characteristics related to size and tissue penetrability. There have been some approved anti-angiogenic therapies for ophthalmic conditions, not related to vNAR. This includes biologics and chimeric proteins that neutralize vascular endothelial growth factor (VEGF)165, which are injected intravitreal, causing discomfort and increasing the possibility of infection. In this paper, we present a vNAR antibody against human recombinant VEGF165 (rhVEGF165) that was isolated from an immunized Heterodontus francisci shark. A vNAR called V13, neutralizes VEGF165 cytokine starting at 75 µg/mL in an in vitro assay based on co-culture of normal human dermal fibroblasts (NHDFs) and green fluorescence protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) cells. In the oxygen-induced retinopathy model in C57BL/6:Hsd mice, we demonstrate an endothelial cell count decrease. Further, we demonstrate the intraocular penetration after topical administration of 0.1 µg/mL of vNAR V13 by its detection in aqueous humor in New Zealand rabbits with healthy eyes after 3 h of application. These findings demonstrate the potential of topical application of vNAR V13 as a possible new drug candidate for vascular eye diseases.

Atypical antigen recognition mode of a shark immunoglobulin new antigen receptor (IgNAR) variable domain characterized by humanization and structural analysis.

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.