VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica)

Abstract

The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10−8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning.

Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica)

“…The preparation of the anti-Venus VNAR phage library and purified Venus antigen has been described in a previous report [24]. The input phage library from immunized shark spleen cells was amplified.…”

“…Kim et al immunized banded houndsharks with SARS-CoV-2 spike RBDs to produce antigen-specific VNARs [23]. We immunized Japanese topesharks with the Venus protein and reported an increase in the Venus-specific IgNAR antibody titer [24]. We constructed a VNAR display phage library from immunized sharks, enriched the antigen-specific VNARs under general panning conditions, and isolated several VNAR clones.…”

“…We applied an approach that combines selection pressure and NGS analysis to VNAR screening using phage display technology and verified its usefulness in solving this problem. We used the library produced in a previous study and enriched the VNARs under various selection pressures [24]. The selection pressures examined included conditions that mimicked the unique blood environment of sharks (high salt concentration, high urea concentration, and low temperature) and conditions that leveraged their distinctive physical properties of VNARs (preheating).…”