Bamboo Sharks for Affordable Single Domain Antibody Production

斑竹鯊用於生產經濟適用的單域抗體

Student thesis: Doctoral Thesis

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Award date18 Mar 2021

Abstract

Single domain antibodies (sdAbs) derived from heavy chain only antibody of camelids and sharks attract many interests because their values in drug development and scientific research. However, the farming of sharks for immunization are costly and difficult. Here we demonstrated white-spotted bamboo shark (Chiloscyllium plagiosum) being cultivated readily and commercially to be a shark for high affinity antigen-specific sdAbs generation. We found that bamboo sharks have four clusters for immunoglobulin new antigen receptor (IgNAR), the heavy chain only antibody, in the genome and express both secretory and transmembrane IgNARs in blood. We identified the detailed structural configuration of germline IgNAR clusters and how many IgNAR monomer constitutes multiform IgNAR multimers. Upon immunization with GFP and iRFP713, bamboo sharks develop efficient immune response represented by elevated immune cells and IgNAR titer against antigens. Deep sequencing on the variable domain of IgNAR (vNAR, a type of sdAbs) showed that the diversity of vNAR decreased upon immunization, suggesting the enrichment of specific IgNARs, and characterized the development of CDR3 diversity. The effective immunization program for bamboo sharks is therefore validated. Then we isolated GFP-specific vNARs with affinities at subnanomolar from immunized bamboo shark blood by phage display. And we validated their functions in mammalian cells and constructed bivalent/biparatopic vNARs with picomolar affinities. Moreover, we isolated several anti-SARS-CoV-2 spike protein vNARs from the same phage display library within 17 days. Our study provides a comprehensive picture of IgNAR immune repertoire of bamboo shark and proves that bamboo shark is ideal for high affinity antigen-specific vNARs discovery and development as immunoreagents and diagnostic reagents.

    Research areas

  • single domain antibody, bamboo shark, IgNAR, immunization, vNAR, immune repertoire