title: Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phage-displayed library of macaque origin.
abstract: BACKGROUND
  Botulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic substance known. It causes naturally-occurring food poisoning, and is among the biological agents at the highest risk of being weaponized. Several antibodies neutralizing BoNT/A by targeting its heavy chain (BoNT/A-H) have been isolated in the past. For the first time however, an IgG (4LCA) recently isolated by hybridoma technology and targeting the BoNT/A light chain (BoNT/A-L), was shown to inhibit BoNT/A endopeptidase activity and protect in vivo against BoNT/A. In the present study, a phage-displayed library was constructed from a macaque (Macaca fascicularis) hyper-immunized with BoNTA/L in order to isolate scFvs inhibiting BoNT/A endopeptidase activity for clinical use.
  
  
  RESULTS
  Diversity of the scFvs constituting the library was limited due to the frequent presence, within the genes intended to be part of the library, of restriction sites utilized for its construction. After screening with several rounds of increasing stringency, as is usual with phage technology, the library got overwhelmed by phagemids encoding incomplete scFvs. The screening was successfully re-performed with a single round of high stringency. In particular, one of the isolated scFvs, 2H8, bound BoNT/A1 with a 3.3 nM affinity and effectively inhibited BoNT/A1 endopeptidase activity. The sequence encoding 2H8 was 88% identical to human germline genes and its average G-score was -0.72, quantifying the high human-like quality of 2H8.
  
  
  CONCLUSIONS
  The presence of restrictions sites within many of the sequences that were to be part of the library did not prevent the isolation of an scFv, 2H8, by an adapted panning strategy. ScFv 2H8 inhibited toxin endopeptidase activity in vitro and possessed human-like quality required for clinical development. More generally, the construction and screening of phage-displayed libraries built from hyper-immunized non-human primates is an efficient solution to isolate antibody fragments with therapeutic potential.
authors: Unité de Biotechnologie des Anticorps, et des Toxines, Département de Microbiologie, Institut de Recherche Biomédicale des Armées (IRBA-CRSSA), 24 Avenue des Maquis du Grésivaudan, BP 87, 38702 La Tronche Cedex, France. pthullier@yahoo.com
authors: Siham
authors: S
authors: Chahboun
authors: Michael
authors: M
authors: Hust
authors: Yvonne
authors: Y
authors: Liu
authors: Thibaut
authors: T
authors: Pelat
authors: Sebastian
authors: S
authors: Miethe
authors: Saskia
authors: S
authors: Helmsing
authors: Russell Ga
authors: RG
authors: Jones
authors: Dorothea
authors: D
authors: Sesardic
authors: Philippe
authors: P
authors: Thullier
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors* / Q000276:immunology; D010450:Endopeptidases / Q000276:immunology; D008251:Macaca; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000302:isolation & purification* / Q000494:pharmacology*
chemical_list: D019151:Peptide Library; D057127:Single-Chain Antibodies; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A