Isolation of highly selective IgNAR variable single-domains against a human therapeutic Fc scaffold and their application as tailor-made bioprocessing reagents

Magdalena J Buschhaus* (Corresponding Author), Stefan Becker, Andrew J Porter, Caroline J Barelle

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

The adaptive immune system of cartilaginous fish (Elasmobranchii), comprising of classical hetero-tetrameric antibodies, is enhanced through the presence of a naturally occurring homodimeric antibody-like immunoglobulin-the new antigen receptor (IgNAR). The binding site of the IgNAR variable single-domain (VNAR) offers advantages of reduced size (<1/10th of classical immunoglobulin) and extended binding topographies, making it an ideal candidate for accessing cryptic epitopes otherwise intractable to conventional antibodies. These attributes, coupled with high physicochemical stability and amenability to phage display, facilitate the selection of VNAR binders to challenging targets. Here, we explored the unique attributes of these single domains for potential application as bioprocessing reagents in the development of the SEED-Fc platform, designed to generate therapeutic bispecific antibodies. A panel of unique VNARs specific to the SEED homodimeric (monospecific) 'by-products' were isolated from a shark semi-synthetic VNAR library via phage display. The lead VNAR candidate exhibited low nanomolar affinity and superior selectivity to SEED homodimer, with functionality being retained upon exposure to extreme physicochemical conditions that mimic their applicability as purification agents. Ultimately, this work exemplifies the robustness of the semi-synthetic VNAR platform, the predisposition of the VNAR paratope to recognise novel epitopes and the potential for routine generation of tailor-made VNAR-based bioprocessing reagents.

Original languageEnglish
Pages (from-to)385-399
Number of pages15
JournalProtein engineering, design & selection : PEDS
Volume32
Issue number9
Early online date2 Mar 2020
DOIs
Publication statusE-pub ahead of print - 2 Mar 2020

Keywords

  • affinity ligand
  • phage display
  • SEED bispecific antibody platform
  • shark IgNAR
  • variable new antigen receptor
  • BISPECIFIC ANTIBODIES
  • COMPLEX
  • IMMUNOGLOBULIN
  • ANTIBODY FRAGMENTS
  • ANTIGEN RECEPTOR IGNAR
  • PLATFORM
  • AFFINITY LIGANDS
  • SHARK
  • SEED
  • PURIFICATION

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