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

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.

Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein

“…VNARs offers several advantages over conventional IgG antibodies, including a small size that allows for more efficient tissue penetration, high stability, and specificity for cryptic epitopes [ 18 ]. Since antigen-specific VNARs from immunized libraries generally have higher binding affinities than naïve and synthetic libraries [ 19 ], there is a growing demand for validated animal models that can produce antigen-specific VNARs to broaden the scope of VNAR-based research. In this study, we constructed an immunized library from banded houndshark for the first time to identify VNARs against SARS-CoV-2 RBD.…”

“…A primary reason medical and biotechnological researchers prefer nanobodies is the relatively limited availability of animal models for discovering VNARs [ 19 ]. Although various IgNARs have been discovered in numerous cartilaginous fish species [ 20 ], the six reported SARS-CoV-2 specific VNARs were derived exclusively from the nurse shark (Ginglymostoma cirratum) [ 11 , 12 , 16 ], white-spotted bamboo shark (Chiloscyllium plagiosum) [ 13 , 14 ], and a synthetic library [ 15 ].…”

“…Banded houndsharks frequently inhabit the coastal waters of Korea [ 21 ], pose no threat to humans, and adapt well to captivity [ 22 , 23 ]. Interestingly, immunization of banded houndsharks has not been previously reported [ 19 ], although their IgNARs have been identified [ [24] , [25] , [26] ]. In this study, we isolated several SARS-CoV-2 RBD-specific VNARs from the banded houndshark, including CoV 2 NAR-1, which exhibited high binding affinity and neutralizing activity against SARS-CoV-2 pseudoviruses.…”

The influence of antibody humanization on shark variable domain (VNAR) binding site ensembles

“…Structurally, these antibodies are characterized by a conserved tryptophan residue in the CDR1 loop and limited CDR3 loop diversity, however, the functional role of these antibodies remains still elusive. Type IV VNARs, which are primarily found in dogfish sharks, wobbegong, small-spotted catsharks and bamboo sharks contain only the two canonical cysteine residues ( 9 , 13 , 24 , 25 ). In this study, we investigate the consequences and effects of antibody humanization of the spiny dogfish shark VNAR E06 ( Figure 2 ).…”

The specific biopanning of single‐domain antibody against haptens based on a functionalized cryogel

“…In this procedure, specific phages were obtained from the display libraries by biopanning based on the physical linkage between genotype and phenotype 9 . During traditional biopanning, antigens were usually coated on immunotubes or microtiter plates 10‐13 . The specific phages with high affinity were obtained through phage incubation and several rounds of binding, washing, and eluting 14 .…”