Construction of a phage-displayed naïve nurse shark VNAR single domain antibody library and its large diversity revealed by next-generation sequencing analysis

Authors

• H Bian
• M Feng
• X Wu
• T Fu
• J Hong
• B Felming
• M Flajnik
• M Ho

Abstract

Background: The shark VNAR (variable domain of new antigen receptor) antibody is one of the smallest antibodies in the animal kingdom with sizes ranging from 12-15 kDa. With great stability, solubility, and ability to penetrate tissues and bind with hidden functional sites in target proteins, shark VNAR possess distinct advantages over conventional antibodies as novel therapeutics in cancer and other diseases. Methods: With a highly efficient method developed in this study based on PCR- Extension Assembly and Self-Ligation (named ‘EASeL’), we constructed a phage-displayed VNAR antibody library with a size of 1.2 x 1010 from six naïve adult nurse sharks (Ginglymostoma cirratum). The plasmid DNA of this library was extracted for Next Generation Sequencing (NGS) analysis and over 1.19 million full length unique VNAR sequences were analyzed. Results: This naïve shark library is highly diversified because it covers not only all four known VNAR types (types I-IV) but also VNAR sequences that have not been categorized in any known types. The amino acid variability analysis showed the sequence diversity is mostly in the CDR3. CDR3 lengths and cysteine numbers based on NGS analysis showed unique patterns as compared to conventional IgG antibody sequences. VNAR binders targeting cancer and viral antigens were isolated from this library. Furthermore, shark VNAR single domain antibodies have been successfully produced in E.coli and validated for their antigen binding. Conclusion: Our new shark single domain antibody library is a useful platform for selecting therapeutic antibodies target challenging antigens.

Scientific Focus Area: Immunology

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