Towards the improvement in stability of an anti-Aβ single-chain variable fragment, scFv-h3D6, as a way to enhance its therapeutic potential

Laia Montoliu-Gaya, Javier Murciano-Calles, Jose C. Martinez, Sandra Villegas

Research output: Contribution to journalArticleResearchpeer-review

5 Citations (Scopus)

Abstract

© 2017 Informa UK Limited, trading as Taylor & Francis Group. ScFv-h3D6 is a single-chain variable fragment derived from the monoclonal antibody bapineuzumab that prevents Aβ-induced cytotoxicity by capturing Aβ oligomers. The benefits of scFv-h3D6 treatment in Alzheimer’s disease are known at the behavioural, cellular and molecular levels in the 3xTg-AD mouse model. Antibody-based therapeutics are only stable in a limited temperature range, so their benefit in vivo depends on their capability for maintaining the proper fold. Here, we have stabilized the scFv-h3D6 folding by introducing the mutation VH-K64R and combining it with the previously described elongation of the VL domain (C3). The stabilities of the different scFv-h3D6 constructs were calculated from urea and thermal denaturation followed by Trp-fluorescence, CD and DSC and resulted in the order C3 > K64R/C3 > VH-K64R ≥ scFv-h3D6; showing that the combination of both mutations was not additive, instead they partially cancelled each other. The three mutants assayed showed a decreased aggregation tendency but maintained their capability to aggregate in the form of worm-like fibrils, basis of the protective effect of scFv-h3D6. Cytotoxicity assays showed that all the mutants recovered cell viability of Aβ-treated neuroblastoma cell cultures in a dose-dependent manner and with efficiencies that correlated with stability, therefore improving the therapeutic ability of this antibody.
Original languageEnglish
Pages (from-to)167-175
JournalAmyloid
Volume24
Issue number3
DOIs
Publication statusPublished - 3 Jul 2017

Keywords

  • Protein stability
  • aggregation
  • cytotoxicity
  • protein design
  • unfolding

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