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

John Steven, Obinna C. Ubah*, Magdalena Buschhaus, Marina Kovaleva, Laura Ferguson, Andrew J. Porter, Caroline J. Barelle

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter


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.

Original languageEnglish
Title of host publicationGenotype Phenotype Coupling
Subtitle of host publicationMethods in Molecular Biology
EditorsS. Zielonka, S. Krah
Place of PublicationNew York
Number of pages28
ISBN (Electronic)9781493998531
ISBN (Print)9781493998524
Publication statusPublished - 2020

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


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


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