Construction and next-generation sequencing analysis of a large phage-displayed V(NAR) single-domain antibody library from six naïve nurse sharks

Feng, Mingqian; Bian, Hejiao; Wu, Xiaolin; Fu, Tianyun; Fu, Ying; Hong, Jessica; Fleming, Bryan D; Flajnik, Martin F; Ho, Mitchell

Shark V NAR domains may have advantageous properties over conventional IgG. First, sharks are evolutionarily distant from mammals on the phylogenetic tree, therefore can generate high-affinity binders to structurally conservative mammalian drug targets. These may include highly conserved heparan sulfate proteoglycans, G-protein coupled receptors, and ion channels that may exhibit poor immunogenicity in mice and rabbits [15, 17] . Second, the elongated CDR3 in shark and camel antibodies has the ability to seek out buried epitopes and enzyme functional sites [2, 15, 17, 18] . The shark IgNAR CDR3 regions are relatively longer (ranging from 9 to 34 a.a. and including various numbers of cysteine residues) compared to mouse or human counterparts. This can potentially lead to a larger diversity of structures that can interact with more diversified antigens [16] . Similarly, the longer CDR3 region in shark antibodies possesses the extraordinary capacity to form long finger-like extensions that can probe proteins for hidden epitopes [2] . Third, conventional antibodies may have poor tissue penetration ability due to their large size [19] . Whole IgG is 150 kDa and scFv fragments 25-30 kDa. V NAR domains can be as small as 12-15 kDa. Finally, shark V NAR domain antibodies have structural advantages and are easily expressed in Escherichia coli systems [2] . Sharks enrich their blood with urea to maintain osmotic balance in the marine environment, so shark antibody structure has evolved to become particularly stable [4] .