Bamboo Shark as a Small Animal Model for Single Domain Antibody Production.

Likun Wei,Hongming Dong,Yuan Jiang,Naibo Yang,Jiajun Wu,Jinhua Gong,Limin Feng,Leo Lai Chan,Dan Su,Meiniang Wang,M A R Al Azad,Haitao Xiang,Jiahai Shi

doi:10.3389/fbioe.2021.792111

Abstract

The development of shark single domain antibodies (sdAbs) is hindered by the high cost and tediousness of large-sized shark farming. Here, we demonstrated white-spotted bamboo sharks (Chiloscyllium plagiosum) being cultivated commercially as a promising small animal model to produce sdAbs. We found that immunoglobulin new antigen receptor (IgNAR) presented in bamboo shark genome, transcriptome, and plasma. Four complete IgNAR clusters including variable domains (vNARs) were discovered in the germline, and the Variable–Joining pair from IgNAR1 cluster was dominant from immune repertoires in blood. Bamboo sharks developed effective immune responses upon green fluorescent protein (GFP), near-infrared fluorescent protein iRFP713, and Freund’s adjuvant immunization revealed by elevated lymphocyte counts and antigen specific IgNAR. Before and after immunization, the complementarity determining region 3 (CDR3) of IgNAR were the major determinant of IgNAR diversity revealed by 400-bp deep sequencing. To prove that bamboo sharks could produce high-affinity IgNAR, we isolated anti-GFP and anti-iRFP713 vNARs with up to 0.3 and 3.8 nM affinities, respectively, from immunized sharks. Moreover, we constructed biparatopic vNARs with the highest known affinities (20.7 pM) to GFP and validated the functions of anti-GFP vNARs as intrabodies in mammalian cells. Taken together, our study will accelerate the discovery and development of bamboo shark sdAbs for biomedical industry at low cost and easy operation.

Highlights

We discovered that IgNARshort transmembrane form, IgNARlong secretory form, and multiple immunoglobulin new antigen receptor (IgNAR) secretory multimers presented in the blood of bamboo sharks
We discovered 37 IgM clusters distributed on seven chromosomes (Chr) and seven IgNAR clusters located near one end of Chr44 in the white-spotted bamboo shark genome (Figure 1A) (Zhang et al, 2020a)
A conserved cysteine existed at the secretory tail (Sec) carboxyl terminus of all three shark Ig isotypes and human IgA and IgM, which is critical for multimerization (Supplementary Figure S1B) (Smith et al, 2012b; Castro et al, 2013; Mashoof and Criscitiello, 2016)

Summary

Introduction

Single domain antibodies (sdAbs) are preferred over traditional antibodies because of their simple architecture, high thermal and chemical stability, good solubility, high tissue penetration, easy modularity, and straightforward expression in Escherichia coli (Holliger and Hudson, 2005; Simmons et al, 2006; Koenning et al, 2017; Steven et al, 2017; Ubah et al, 2017; Ubah et al, 2020; Stocki et al, 2021). In the same manner as VHH production (Holliger and Hudson, 2005), vNARs from immunized libraries (CamachoVillegas et al, 2013; Kovaleva et al, 2017; Leow et al, 2018) generally have higher affinities and specificities than those from semi-synthetic libraries (Häsler et al, 2016; Cabanillas-Bernal et al, 2019; Stocki et al, 2021) and naïve libraries (Simmons et al, 2006; Feng et al, 2019) This result is because IgNARs have experienced iterative affinity maturation in immunized sharks upon immunization (Dooley et al, 2006). They can be bred artificially and domesticated to eat artificial feed (Chen and Liu, 2006), making them suitable for large-scale husbandry

Methods

Results

Conclusion