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

Abstract

BackgroundShark new antigen receptor variable domain (VNAR) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies.MethodsHere, 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).ResultsThe 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.ConclusionsThe 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.

Antibody Structure and Function: The Basis for Engineering Therapeutics

“…The development of libraries for selection has expanded the utility of generating potent molecules [312,313]. As with nanobodies, these molecules can be engineered to be more human-like and have been used to isolate binders to CXCR4, a druggable GPCR [314,315], HER2, PD1, and glypican 3 [316].…”

Landscape of Non-canonical Cysteines in Human VH Repertoire Revealed by Immunogenetic Analysis

“…However, non-canonical cysteines are normally encoded by certain human diversity gene (D gene) segments, mainly IGHD2 and other D gene families. Non-canonical cysteines are thought to be less prevalent in human compared with species such as chicken (Wu et al, 2012), camel (Muyldermans et al, 1994), llama (Harmsen et al, 2000), shark (Feng et al, 2019;Stanfield et al, 2004), and cow (Haakenson et al, 2019;Saini et al, 1999;Wang et al, 2013). Non-canonical cysteines in these non-human species form various intra-heavy-chain complementarity-determining region 3 (CDR-H3) disulfide bonds and disulfide bonds between CDR-H3 and other CDRs or with framework regions (FRs).…”

Diagnostic and therapeutic potential of shark variable new antigen receptor (VNAR) single domain antibody

“…From this naïve nurse shark VNAR library, a panel of VNAR binders that specific to cancer therapy-related antigens, which are glypican-3 (GPC3), human epidermal growth factor receptor 2 (HER2) and programmed cell death-1 (PD1) were isolated. Meanwhile, VNARs specific to viral antigens, such as the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) spike proteins were also isolated recently [52]. Therefore, the findings obtained herein have convinced where VNAR clones selected from the naïve nurse shark VNAR library may potentially be developed as therapeutic antibodies after humanization process undertaken.…”

“…However, such method is time consuming, and may experience a low efficiency in the ligation and transformation step. Hence, a group of researchers had developed a library construction method based on PCR extension assembly followed by self-ligation (EASeL), which significantly shortened the time needed to construct the library [52]. Using this method, a large and highly diverse VNAR library derived from six antigen-naïve nurse sharks (Ginglymostoma cirratum), with a library size of 10 10 has been recently generated.…”