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

Abstract

ABSTRACT Background Shark new antigen receptor variable domain (VNAR) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies. Methods Here, we developed a library construction method based on polymerase chain reaction (PCR)-Extension Assembly and Self-Ligation (named “EASeL”) to construct a large VNAR antibody library with a size of 1.2 × 1010 from six naïve adult nurse sharks (Ginglymostoma cirratum). Results The next-generation sequencing analysis of 1.19 million full-length VNARs revealed that this library is highly diversified because it covers all four classical VNAR types (Types I–IV) including 11% of classical Type I and 57% of classical Type II. About 30% of the total VNARs could not be categorized as any of the classical types. The high variability of complementarity determining region (CDR) 3 length and cysteine numbers are important for the diversity of VNARs. To validate the use of the shark VNAR library for antibody discovery, we isolated a panel of VNAR phage binders to cancer therapy-related antigens, including glypican-3, human epidermal growth factor receptor 2 (HER2), and programmed cell death-1 (PD1). Additionally, we identified binders to viral antigens that included the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) spike proteins. The isolated shark single-domain antibodies including Type I and Type II VNARs were produced in Escherichia coli and validated for their antigen binding. A Type II VNAR (PE38-B6) has a high affinity (Kd = 10.1 nM) for its antigen. Conclusions The naïve nurse shark VNAR library is a useful source for isolating single-domain antibodies to a wide range of antigens. The EASeL method may be applicable to the construction of other large diversity gene expression libraries.