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Antibody Therapeutics logoLink to Antibody Therapeutics
. 2020 Jan 21;3(1):1–9. doi: 10.1093/abt/tbaa001

Ancient species offers contemporary therapeutics: an update on shark VNAR single domain antibody sequences, phage libraries and potential clinical applications

Hejiao English 1,2, Jessica Hong 1,2, Mitchell Ho 1,2,
PMCID: PMC7034638  PMID: 32118195

ABSTRACT

The antigen binding variable domain (VNAR) of the shark immunoglobulin new antigen receptor (IgNAR) evolved approximately 500 million years ago and it is one of the smallest antibody fragments in the animal kingdom with sizes of 12–15 kDa. This review discusses the current knowledge of the shark VNAR single domain sequences and ongoing development of shark VNARs as research tools as well as potential therapeutics, in particular highlighting the recent next-generation sequencing analysis of 1.2 million shark VNAR sequences and construction of a large phage displayed shark VNAR library from six naïve adult nurse sharks (Ginglymostoma cirratum). The large phage-displayed VNAR single domain library covers all the four known VNAR types (Types I–IV) and many previously unknown types. Ongoing preclinical development will help define the utility of shark VNAR single domains as a potentially new family of drug candidates for treating cancer and other human diseases.

Keywords: nurse shark (Ginglymostoma cirratum), phage display library, VNAR single domain, next-generation sequencing, antibody engineering


Statement of Significance: This review discusses recent progress on next-generation sequencing analysis and construction of a large nurse shark VNAR single domain antibody library, the current development of shark VNAR single domain antibodies as research tools, as well as potential therapeutics for treating cancer and other human diseases.

INTRODUCTION

Currently, there are over 400 species of sharks living in the marine ecosystem on our planet [1–3]. Their sizes can vary from 7-inch dwarf lanternshark (Etmopterus perryi) to 39-foot whale shark (Rhincodon typus) [4]. Sharks initially dominated the ocean as top predators about 500 million years ago [1, 4–6]. During that time, their immune system, the earliest adaptive immunity to our knowledge, has evolved to produce the comparable fundamental factors including T cells, B cells and major histocompatibility complexes (MHCs) that are found in mammals [1, 4, 7]. However, there are certain unique immunological features in sharks that are not normally present in humans or other mammals, with the exception of camelids [2, 8]. Sharks began to produce a homodimeric heavy chain-only antibody known as immunoglobulin new antigen receptor (IgNAR), a homodimeric protein composed of heavy chains with an antigen-binding region at the end of each H chain [9] to combat foreign antigens [1, 4, 6, 10, 11] (Fig. 1). The basic Ig fold of modern antibodies is present in the evolutionary ancient shark IgNAR domains even with low sequence conservation [6, 10]. The antigen binding variable domain of these IgNAR (VNAR) is much smaller (12–15 kDa) than classical IgG VH + VL (Fv 25–30 kDa) [7]. The strikingly high homology between VNAR and shark TCR NAR has raised interests in the possible origin of VNAR [6, 10]. It is speculated that VNAR originated from a common NAR domain that was used by both TCR and IgNAR as an antigen binding domain over 450 million years ago and into VNAR or NAR-TCRV to allow improved specialization binding ability for both domain types [6]. In addition, an alternative theory suggests that both NAR-TCR and VNAR may have originated (independently or not) from cell adhesion molecules [5, 12]. Although the origin of IgNAR remains unsolved, the fact that VNAR also exists as an antigen-recognition domain in shark TCRs suggests that VNAR functionally may play a role in both B-cell and T-cell immune responses [1].

Figure 1.

Figure 1

Schematic depiction of human IgG, shark IgNAR and VNAR structures.

Based on the number and position of cysteines, shark VNARs were categorized into four classical types in previous studies [8, 9, 13]. A recent next-generation sequencing (NGS) on Illumina MiSeq analyzed with custom Perl Scripts revealed approximately 1.2 million full-length shark VNAR sequences from a phage-displayed VNAR antibody library constructed from six naïve adult nurse sharks (Ginglymostoma cirratum) [14]. It is the largest scale shark VNAR sequence analysis reported thus far. The NGS analysis reveals that a number of VNAR sequences do not belong to any of the four classical types [15]. As described in Fig. 2, the presence of two canonical cysteines located at both amino acid 21 and 82 are used as a key criterion for us to define Type I–IV VNARs in our NGS analysis. The sequences that do not contain one or both of these cysteines are considered as other types (~5% of the total VNARs analyzed by NGS). The sequences that have both 21C and 82C are further categorized based on their placement of additional cysteines. Type IV VNARs contain only two canonical cysteines found at positions 21 and 82. We further defined Type I VNARs as those contain an extra cysteine at position 34 (21C, 34C, 82C; ~ 24% of the total nurse shark VNARs). This group can be further divided into subtypes based on how many additional cysteines they contain. The classical Type I VNAR as a subtype has a total of six cysteines (~ 11% of the total nurse shark VNARs). Type II and III VNARs have at least one extra cysteine at amino acid 28 (21C, 28C, 82C; 70% of the total VNARs). Among them, ~ 57% of the total VNARs are classical Type II, a subtype with a total of four cysteines. Importantly, approximately 30% of VNARs do not belong to any of the four classical types. The structure and function of these previously unidentified VNAR types require further structure and functional investigation. Due to their high stability, solubility and potential to penetrate tissues and bind to hidden functional sites in target proteins, shark VNARs may potentially possess distinct advantages over conventional antibodies as novel therapeutics for human diseases [3, 8, 16]. We previously compared the known structures of a nurse shark VNAR (PDB 1T6V) [17] to a conventional Fv antibody (PDB 2EIZ) [18] in their complexes with lysozyme, describing the shark VNAR single domain, not the Fv antibody domain, can reach to the buried substrate binding pocket of lysozyme [3]. Based on this observation and other findings targeting virus particles [19, 20] and toxins [21], we have postulated that VNAR single domains may have potential to reach buried functional cavities or grooves with the extended CDR3 loop. Currently, there are several promising shark VNARs in pre-clinical development aiming to be evaluated in clinical trials as therapeutics in humans [8, 16, 22–26].

Figure 2.

Figure 2

Shark VNAR Types I-IV based on the recent NGS analysis of 1.2 million adult nurse shark VNAR sequences. The two canonical cysteines (21C and 82C) are in white circles, whereas the extra cysteines are in black circles. The variable regions are marked as CDR1, HV2, HV4 and CDR3. All the disulfide bridges are shown using connected black lines. Type I (21C, 34C and 82C) and Type II (21C, 28C and 82C) VNARs contain three canonical cysteines as indicated. Subtypes in Type I and Type II VNARs contain 3–9 cysteines.

THE EVOLUTIONARY ORIGINS OF SHARK VNARs

The diversification of the vertebrate adaptive immune system is estimated to have happened over 500 million years ago [10]. Cartilaginous fish such as sharks, skates and rays diverged from the bony fish about 450 million years ago and started to produce immunoglobulin-based antibodies—IgNARs [6, 10]. Previous studies have demonstrated that IgNARs are part of the adaptive immune armory and they have the highest potential for antigen-driven affinity maturation as compared to other Ig types in sharks such as IgW and IgM [6, 11]. In-depth analysis of the evolution of fish and shark immunoglobulin systems has been reviewed by others [5, 12].

VNAR phage-displayed libraries and functional validation of a panel of single domain VNAR binders showed that dimerization is not required for high-affinity antigen binding [8, 14, 21, 27, 28]. These observations may suggest that ancient VNARs could be a functional single domain antigen binding domain versus the homodimeric format of IgNAR in modern sharks [6]. This differentiates shark VNAR from camelid VHH (variable domain of camelid heavy chain antibody) evolutionarily, as camelid VHH might evolve from a conventional IgG by simultaneous loss of the light chain and the CH1 domain of heavy chain [10]. However, in our study and others the selection of phage-displayed antibody fragments is an artificial selection system. It is not a biological system to either prove or disapprove whether a single domain can be automated and independently folded in animals.

Sequence homology analysis revealed that VNARs might have evolved from an ancestral antigen receptor or cell adhesion molecule that is incorporated in both IgNAR and shark NAR-TCR as antigen binding domains [6]. Whether modern VNAR might possess more TCRV-like properties such as the ability to bind with MHC-associated peptide complexes with high specificity should be explored and validated in future structural and functional studies. Cell surface proteins and secreted proteins account for only a small portion of tumor antigens. Intracellular proteins are degraded by the proteasome to produce short peptides, some of which are ultimately presented at the cell surface as the MHC-associated peptide complexes that can be recognized by TCRs on T cells [29]. The similarity in amino acid sequences might enable a phage-displayed VNAR single domain library to be used as a platform to select TCR-mimic antibodies, which can be used to bind intracellular cancer antigens by targeting MHC-associated peptide complexes.

UPDATES ON SHARK VNAR SEQUENCES AND TYPES

The two β sheets of VNAR domains are held together by two canonical cysteine residues (21C and 82C) in framework regions (FR) 1 and 3. The canonical cysteines can stabilize the immunoglobulin fold via a disulfide bond. In addition to these canonical cysteines that are highly conserved across animals, the complementarity-determining region 3 (CDR3), CDR1 and certain FR can have additional cysteines that form disulfide bonds within the V domain [1, 2, 30]. Based on the numbers and positions of these extra cysteines in the VNAR domain, IgNARs are classified into four types including classical [15] and non-classical subtypes (Fig. 2). As the sequence diversity of VNAR is primarily in CDR3, the diversity of cysteine numbers and locations is crucial for VNAR structural diversity [14].

Classical Type I VNAR contains two non-canonical cysteine residues in CDR3, which form two extra disulfide bonds with FR2 (34C) and FR4 to make a relatively rigid antigen binding surface based on the crystal structure of a Type I shark VNAR in complex with lysozyme [17]. Type I was initially reported only in the nurse shark (G. cirratum) [15, 17]. More recently, Type I VNARs were also found in the wobbegong shark (Orectolobus ornatus) [26]. Our group has isolated a Type I VNAR binder from a phage displayed nurse shark single domain VNAR library, which binds to a MERS spike protein [14] The soluble monomeric single domain protein derived from the phage binder, MERS A8, was produced in Escherichia coli and the binding affinity was conserved when produced and folded in a prokaryotic system with a yield of 3 mg purified Type I VNAR single domain protein from 600 mL E. coli culture, while the yield of a Type II VNAR single domain protein was 9 mg from the same volume of culture [14].

Classical Type II VNAR domains have two extra cysteines located in CDR3 and CDR1 (28C), which form a disulfide bond between CDR1 and CDR3. Both positions of the cysteine in CDR3 and the length of CDR3 have expanded the structural diversity of antigen binding surfaces for Type II VNAR domains [28, 31, 32]. Both Type I and II VNARs can have long fingerlike CDR3s that reach into and bind with recessive epitopes [7, 11]. While CDR3 in humans generally comprise of 8–12 amino acids, VNARs can have up to 34 amino acids as previously reported [1]. Our recent NGS analysis of over one million nurse shark VNAR sequences demonstrates the CDR3 length of both Type I and Type II VNARs varies greatly between 0 and 40 amino acids with a median CDR3 length of about 20 amino acids [14]. VNARs lack conventional CDR2 which are present in another classical IgG and camelid VHH. Instead, VNARs have two shorter CDR2-like regions: hypervariable region 2 (HV2) and hypervariable region 4 (HV4) separated by a FR [28, 32].

Classical Type III VNARs are similar to Type II VNARs, except there is a highly conserved tryptophan residue in CDR1 after the cysteine. The CDR3 is not as diverse in Type III since Type III VNAR domains are predominantly found in neonatal sharks [8]. Classical Type IV VNARs domains only have two canonical cysteines (21C and 82C) that hold the VNAR together and have the highest similarity to VH domains in conventional IgG found in other species including humans.

Currently, there are several shark VNAR libraries reported using various shark species: banded wobbegong shark [26], spiny dogfish (Squalus acanthias) [27], smooth dogfish (Mustelus canis) [21], bamboo shark (Chiloscyllium plagiosum) [33], horn shark (Heterodontus francisci) [24], banded houndshark (Triakis scyllium) [34] and nurse shark [14, 35, 36] (Table 1). Most of them are either produced from an immunized library or a semi-synthetic library with a diversity ranging from 106 to 109. A phage-displayed VNAR single domain library was generated from nurse sharks (G. cirratum) by immunization of the model antigen lysozyme [36]. A semi-synthetic phage displayed VNAR library derived from wobbegong shark (Orectolobus maculatus) with a diversity of 3 × 107 [37] and expanded to 4 × 108 [38] was constructed by randomizing the CDR3 region [37]. A naïve library previously reported was relatively small (1 × 107) from naïve adult spiny dogfish (S. acanthias) and smooth dogfish (M. canis) sharks [21]. The naïve VNAR libraries derived spiny dogfish and smooth dogfish sharks have no Type I VNAR [21]. The limitations in the library size and types of VNARs may be the major reasons why high affinity VNARs is generally difficult to isolate from a naïve shark single domain library. The naïve nurse shark VNAR phage-displayed library that we constructed from six nurse sharks is currently the largest shark single domain library with a diversity of 1.2 × 1010 containing all four known classical types of VNAR, including 11% of classical Type I, 57% of classical Type II and about 30% of the VNARs that do not belong to any of the classical VNAR types (Table 1), making it a unique tool to potentially isolate high affinity antibody candidates compared to other libraries. To construct a large antibody phage library, we developed a library construction method based on polymerase chain reaction (PCR)-Extension Assembly and Self-Ligation (named “EASeL”) [14]. Interestingly, this library includes VNARs from shark B cells and shark T cells. According to the NGS analysis results of 1.2 million full-length sequences, most of the VNAR sequences in this library are Type I and Type II [14]. Interestingly, over 56 000 of the nurse shark VNAR sequences in our library (~5% of the total nurse shark VNARs) cannot be categorized in any of the four known VNAR types due to variability in cysteine numbers and locations. A significant percentage of previously undescribed types of VNARs merit further investigation [14]. Traditionally, phage displayed antibody library diversity is analyzed by isolation of dozens or hundreds of clones for Sanger sequencing. Modern NGS approaches can be used to assess library diversity by sequencing over 106 clones [39]. The diversity of the new shark VNAR library was analyzed by NGS, revealing 85% (over 1 million) of the 1.2 million sequences appeared only once [14]. The unparalleled repertoire of unique locations and numbers of cysteines may produce a large variety of loop structures that recognize a wide range of epitopes.

Table 1.

Summary of shark VNAR single domain libraries

Library type Inventors Diversity Shark species Library type Binder types Antigens of binders isolated from library Reference
Phage Commonwealth Scientific and Industrial Research Organization (CSIRO, Australia) 3 × 108 O. maculatus Semi-synthetic (CDR3 randomization) Type I and II Human periodontal disease (Kgp, HRgp), AMA1, Tom70 Nuttall et al., 2002; 2003; 2004 [38, 58, 59]
Phage University of Aberdeen (UK) 5 × 106 G. cirratum Immunized Types I and II Hen egg-white lysozyme Dooley et al., 2003 [36]
Phage Naval Research Laboratory (US) 1 × 109 S. acanthias Semi-synthetic (CDR3 randomization) Type II and III Ricin, SEB, BoNT/A toxoid Liu et al., 2007 [27]
Phage Naval Research Laboratory (US) 1 × 107 M. canis Naïve Type II and III Cholera toxin Liu et al., 2007 [21]
Phage Tokyo University of Marine Science and Technology (Japan) 3.7 × 107 T. scyllium Immunized Unknown Hen egg white lysozyme Otani et al., 2012 [34]
Phage CICESE (Center for Scientific Research and Higher Education at Ensenada, US) 1.2 × 109 H. francisci Immunized Unknown TNFα Camacho-Villegas et al., 2013 [24]
Yeast Technische Universität Darmstadt (Germany) 2 × 108 C. plagiosum Semi-synthetic (CDR3 randomization) Unknown EpCAM, HTRA1, and EphA2 Zielonka et al., 2014 [28]
Phage Elasmogen (UK) 108 G. cirratum Immunized Unknown Human and mouse Induced costimulatory ligand Kovaleva et al., 2017 [35]
Phage Universiti Sains Malaysia (Malaysia) 1.16 × 106 O. ornatus Immunized Type I and II Malaria Leow et al., 2018 [26]
Phage National Cancer Institute (NCI) (US) 1.2 × 1010 G. cirratum Naive Type I-IV GPC3, HER2, PD1, MERS, SARS, P. exotoxin Feng et al., 2019 [14]

A summary of shark VNAR single domain libraries produced by academic institutes and commercial companies.

Previous literature showed sequence variability in immunized sharks in CDR1, HV2 and HV4 [28, 32, 40]. However, the NGS analysis of naïve VNAR sequences showed minimal sequence variability in CDR1, HV2 and HV4. The diversity of the naïve VNARs appears to be mostly in CDR3 sequences, while CDR1, HV2 and HV4 likely acquire more sequence diversity during antigen-driven affinity maturation [14]. This finding suggests that increasing sequence diversity in CDR1, HV2 and HV4 through random mutagenesis could potentially mimic in vivo affinity maturation process in sharks and yield high affinity functional binders [41].

POTENTIAL CLINICAL APPLICATION FOR SHARK VNARS

Shark VNAR domains possess unique features and advantages in potential clinical application compared to conventional IgG, including smaller sizes, modifiable half-life, higher tissue penetration ability [25], higher solubility and stability [42] and potential to protrude in buried functional sites in antigens [43]. Single domain antibodies, including camel VHHs [44, 45] and human VH single domains [46–48], have the potential to bind the hidden clefts or grooves in antigens to block the receptor/ligand interactions [17, 20, 49, 50]. On the other hand, the potential immunogenicity of shark antibodies could limit their clinical applications including chimeric antigen receptor T cells. To address this potential issue, a study was conducted to humanize the framework of VNAR by grafting antigen interacting regions to a human framework [51]. The humanized VNAR maintained part of the antigen binding; however, more structure and computational studies are needed for more effective humanization approaches. Furthermore, VNARs have been isolated against vascular endothelial growth factor (VEGF) for treating uveitis, diabetic retinopathies and age-related macular degeneration (AMD) [25, 35]. The designed macular route of delivery differentiated the unique features of VNARs [25, 35].

Currently, several academic institutions and companies are developing naïve, immunized, or synthetic VNAR as potential therapeutics for human diseases (Table 2). The Commonwealth Scientific and Industrial Research Organization (CSIRO) in Australia described semi-synthetic and naïve shark libraries and isolated various VNARs against apical membrane antigen 1 (AMA1) in malaria [20]. Elasmogen Ltd developed several VNARs from immunized, synthetic and naïve libraries targeting inducible T cell costimulatory ligand (ICOSL) and tumor necrosis factor-alpha (TNF-α) [52, 53]. Angiogenesis plays an important role in many human diseases, including eye diseases, by stimulating the development of therapeutic agents that target the pathological angiogenic process in eyes. Bevacizumab is a humanized mouse monoclonal antibody targeting human VEGF for treating cancers. It has been used off-label to treat a specific eye disease called AMD, the leading cause of age-related irreversible blindness. A smaller Fab fragment (48 kDa) of bevacizumab (called ranibizumab) has been approved for treating AMD based on clinical trials. Although Ranibizumab stabilizes and improves vision in over 90% of patients [54], it is administered by intravitreal injection, causing discomfort and increasing the possibility of infection. Recently, a head-to-head comparison of bevacizumab and ranibizumab showed both drugs have equivalent effects clinically [55]. Interestingly, a shark VNAR (named V13) with a long CDR3 (27 amino acids) against (VEGF) was isolated from a male H. francisci shark immunized with recombinant human VEGF. The group administered VNAR V13 to rabbits with healthy eyes and showed intraocular penetration, indicating the superior tissue penetration ability of VNARs [25]. The finding may support the use of shark VNARs as an eye drop for human eye diseases. Further studies will be needed to compare the small size VNARs (e.g. V13) with classical antibodies (e.g. bevacizumab and ranibizumab) for treating eye diseases and other human diseases that require tissue penetration. Our lab has used the new large shark VNAR library to isolate a panel of shark single domain antibodies that bind tumor antigens (e.g. GPC3, Her2) and pathogens (e.g. Pseudomonas aeruginosa, MERS, SARS) [14]. We have isolated a Type II shark VNAR single domain (named B6) which binds specifically to Pseudomonas exotoxin with high affinity (KD = 10 nM). We established a protocol to quickly produce Type I and Type II shark VNAR single domain protein in E. coli for functional analysis [14]. The Type II B6 shark VNAR single domain can be expressed and purified from E. coli culture and has the ability to inhibit P. exotoxin activity. Ongoing studies at the NCI are validating the therapeutic potential of these VNARs and other binders isolated from our nurse shark VNAR library.

Table 2.

Preclinical development of shark VNARs

Name Target antigen Shark species Library type Application Developmental stage Inventors Reference
14I-1, 14I1-M15 AMA1 O. Maculatus Naïve and synthetic Malaria Lead identification CSIRO Health Sciences and Nutrition (Australia) Henderson et al., 2007 [20]
Anti-TNF vNAR TNF H. francisci Repeated immunized Endotoxic shock Pre-clinical Institute Nacional de Cardiologia Ignacio Chavez (Mexico) Bojalil et al., 2013 [22]
BFF1 BAFF G. Cirratum Semi-synthetic (CDR3 randomization) Multiple sclerosis Lead identification Ossianix (UK) Hasler et al., 2016 [60]
vNAR-D01 Aurora-A kinase O. Maculatus Synthetic Solid tumors Lead identification University of Leeds (UK) Burgess et al., 2016 [23]
ELN/21, ELN/22 ICOSL G. Cirratum Immunized, naïve, and synthetic Auto-immune disease, uveitis In vivo validation Elasmogen (UK) Kovaleva et al., 2017 [35]
D1-BA11-C4, D1-Fc-C4 TNF-α G. Cirratum Immunized Polyarthritis Pre-clinical Elasmogen (UK) Ubah et al., 2017; 2019 [52, 53]
V13 VEGF H. francisci immunized Vascular eye disease Lead identification CONACYT (Mexico) Camacho-Villegas et al., 2018 [25]
D1-BA11-C4, D1-Fc-C4 TNF-α G. Cirratum Immunized Polyarthritis Pre-clinical Elasmogen (UK) Ubah et al., 2019 [52]
F1 GPC3 G. Cirratum Naïve Liver Cancer Lead identification NCI (US) Feng et al., 2019 [14]
Her2A6 Her2 G. Cirratum Naïve Her2 positive solid tumors Lead identification NCI (US) Feng et al., 2019 [14]
PD1A1 PD1 G. Cirratum Naïve Solid tumors Lead identification NCI (US) Feng et al., 2019 [14]
MERS A3, A7, A8, B4, B5 MERS spike protein G. Cirratum Naïve MERS virus Lead identification NCI (US) Feng et al., 2019 [14]
SARS binder SARS spike protein G. Cirratum Naïve SARS virus Lead identification NCI (US) Feng et al., 2019 [14]
PE38 B6 P. exotoxin G. Cirratum Naïve Pseudomonas infection Lead identification NCI (US) Feng et al., 2019 [14]

The VNAR single domains that are being developed by commercial companies and academic institutes for potential clinical applications are listed.

Once immunogenicity issues for therapeutic VNARs are further addressed by improving humanization approaches for VNAR and identifying an appropriate, fully human framework for CDR loop grafting from shark VNARs, VNARs can complement huge unmet market needs for conventional IgG due to their unique advantages [56]. Inhalable, macular, or topical administrable therapeutic VNARs will expand the utility fields for antibodies, and the ability to penetrate tissues and solid tumors will enable VNARs to reach organs that are inaccessible to conventional 150 kDa IgGs [16].

FUTURE DIRECTIONS

Structural, computational and functional research is needed to fully understand the vast sequence and structure diversity of shark VNARs and develop them as a novel family of therapeutics for various clinical applications. With the new report about the white shark genome [57], a comprehensive comparative genome and VNAR sequence analysis of white shark, whale shark, nurse shark and other sharks would be intriguing to gain insight on the origin and diversity of shark VNARs in these ancient fishes that are known to have the earliest adaptive immune system during evolution, and with many of them are still living on the planet today among us. Shark VNARs may function as ancient antigen-recognition domains in shark TCRs and the new large nurse shark phage library that contains VNARs from both shark B and T cells may provide a unique source of TCR-mimic single domains targeting MHC-peptide complexes for treating cancer and other diseases.

ACKNOWLEDGEMENTS

This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (NCI CCR). The construction of large phage displayed nurse shark VNAR single domain antibody libraries and the next-generation sequencing analysis of nurse shark VNARs was funded by the NCI CCR FLEX Intramural Program Technology Development Award to M.H. We thank Dr. Martin F. Flajnik (University of Maryland School of Medicine) for expert advice and providing the buffy coat collected from nurse sharks for our construction of a large phage displayed shark VNAR library at the NCI. We also thank NIH Fellows Editorial Board and NIH Library Editing Service for editorial assistance. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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