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In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties

  • John Steven
  • Obinna C. UbahEmail author
  • Magdalena Buschhaus
  • Marina Kovaleva
  • Laura Ferguson
  • Andrew J. Porter
  • Caroline J. Barelle
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)

Abstract

VNAR domains are the binding regions of new antigen receptor proteins (IgNAR) which are unique to sharks, skates, and rays (Elasmobranchii). Individual VNAR domains can bind antigens independently and are the smallest reported adaptive immune recognition entities in the vertebrate kingdom. Sharing limited sequence homology with human immunoglobulin domains, their development and use as biotherapeutic agents require that they be humanized to minimize their potential immunogenicity. Efforts to humanize a human serum albumin (HSA)-specific VNAR, E06, resulted in protein molecules that initially had undesirable biophysical properties or reduced affinity for cognate antigen. Two lead humanized anti-HSA clones, v1.10 and v2.4, were subjected to a process of random mutagenesis using error-prone PCR. The mutated sequences for each humanized VNAR variant were screened for improvements in affinity for HSA and biophysical properties, achieved without a predicted increase in overall immunogenicity.

Key words

Variable new antigen receptors (VNARs) Humanization Immunogenicity Phage display Error-prone PCR Human kappa light chain 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • John Steven
    • 1
  • Obinna C. Ubah
    • 1
    Email author
  • Magdalena Buschhaus
    • 1
  • Marina Kovaleva
    • 1
  • Laura Ferguson
    • 1
  • Andrew J. Porter
    • 1
    • 2
  • Caroline J. Barelle
    • 1
  1. 1.Elasmogen Ltd.AberdeenUK
  2. 2.Scottish Biologics Facility, School of Medical SciencesUniversity of AberdeenAberdeenUK

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