Dissection of the IgNAR V Domain: Molecular Scanning and Orthologue Database Mining Define Novel IgNAR Hallmarks and Affinity Maturation Mechanisms

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