Discovery of IgNAR

In 1995, the presence of novel Immunoglobulin new antigen receptor (IgNAR) isotype was first identified in the serum of the nurse shark (Ginglymostoma cirratum) by Martin Flajnik and colleagues, in which a single domain solely mediates antigen binding, referred to as V_NAR. IgNAR is a homodimer of heavy chains devoid of light chains and different from previously identified conventional hetero-tetrameric immunoglobulins composed of heavy and light chains. It contains two identical heavy chains (HCs) that are composed of one N-terminal variable domain (V) and five constant domains (C1-C5) that are homologous to IgW constant domains. Similar to camelid antibodies, IgNARs are devoid of light chains (LCs), which is an example of convergent evolution.

Different types of V_NARs

Based upon the number and pattern of noncanonical cysteine residues, which are typically not found in classical variable domains, the IgNAR V domain (V_NAR) molecules have been categorized into four different types.

• Type I V_NARs

Type I variable domains carry extra framework cysteines in framework regions 2 (FR2) and framework regions 4 (FR4), as well as paired cysteines within the extended complementarity-determining regions 3 (CDR3) loops to form disulfide-bonds. Thus far, type I variable domains of IgNAR have only been identified in the nurse shark, Ginglymostoma cirratum.

• Type II V_NARs

Type II V_NARs have a more straightforward pairing of cysteine residues that differ from type I V_NARs. Utilizing an additional cysteine in the CDR1 loop and a similar residue within the CDR3, respectively, resulting in an intramolecular disulfide bond that brings both loops in close vicinity. The function of these various additional disulfide linkages is the stabilization of the extended CDR3 loops, providing a degree of rigidity to what would otherwise be fairly flexible structures. To date, type II IgNAR antibodies have been identified in all shark species studied.

• Type III V_NARs

Type III V_NARs are identified during an investigation into the early IgNAR immune repertoire, which is expressed in all neonatal tissues at high frequencies. Similar to type II domains, type III V_NARs is characterized by an additional cysteine in CDR1 and CDR3, respectively. However, type III domains comprise a limited variability in the CDR regions in terms of amino acid composition and length as well as sequence heterogeneity. This type of V_NARs represents an early broad-spectrum antibody and evolves as a consequence of exposure to protection against a common pathogen in the early development of sharks.

• Type IV V_NARs

Type IV V_NARs differ from all described V_NARs types because they lack non-canonical disulfide bonds as found in all other V_NAR types. It is similar to type III but type IV domains are more flexible with an invariant tryptophan residue in CDR1.

Structure and Features of V_NARs

The structural model of the complete IgNAR molecule shows that C1 and C3 domains of each chain within the molecule cause the dimerization of IgNAR. A small angle between both C3 domains induces the formation of a narrow stalk for the IgNAR. The disulfide-bridged linker connects domains C3 and C4 to induce the flexibility of the stalk. Although the lack of a canonical hinge region, the V_NARs are spaced sufficiently wide for multiple binding epitopes. The V_NARs lack a conventional CDR2 contrary to mammalian variable domains and achieve diversity primarily through CDR3 with additional variation in CDR1 and two further named hypervariable domains (HV) 2 and 4, respectively. It is the variable domains and their regions of diversity (CDR1, HV2, HV4, and CDR3) that primarily define target specificity in these animals.

In typical VH-VL antibodies, the interdomain interface is predominantly hydrophobic, suited to a buried surface. In contrast, these regions in the V_NARs are frequently replaced by a conserved and crisscrossing polar or charged residues (residues Glu46; Lys82; Gln84; Arg101; Lys104), which provides both a hydrophilic face to the surrounding medium. Moreover, the different types of V_NAR CDR loops are stabilized by a particular noncanonical disulfide linkage. The extended protruding CDR3 loop structures of V_NARs appear ideal conformations conducive to binding into cryptic and cleft epitopes, such as enzyme active sites and cell surface proteins.

Development Potentials of V_NARs

As described above, shark IgNARs differ greatly from conventional antibodies in many respects. With a molecular mass of ~12 kDa, the V_NAR domain is the smallest antibody-like antigen binding domain to date. Further beneficial attributes of the V_NAR domain comprise its surprisingly high affinities against antigens, superior stability, tolerance to irreversible thermal denaturation as well as cryptic epitope recognition inaccessible and distinct from conventional antibodies. Additionally, there are multiple opportunities to reformat and functionalize the V_NAR domain, including pH-dependent binding behavior, bispecific binding, multimeric constructs, as well as Fc-based formats, clearly demonstrating the possibility of utilizing those molecules for a plethora of different applications. Owing to their inherent favorable attributes, V_NARs emerge as promising target-binding candidates that become viable therapeutic alternatives for biotechnological, biomedical, diagnostic, and therapeutic applications.

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