title: Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E.
abstract: BACKGROUND
  Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure.
  
  
  RESULTS
  In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay.
  
  
  CONCLUSIONS
  These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development.
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Miethe
authors.affiliation: Division of Bacteriology, National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.
authors.forename: Christine
authors.initials: C
authors.lastname: Rasetti-Escargueil
authors.affiliation: Institut de Recherche Biomédicale des Armées (IRBA-CRSSA), Département de Microbiologie, Unité de biotechnologie des anticorps et des toxines, 24 avenue des Maquis du Grésivaudan, B.P. 87, 38702 La Tronche Cedex, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Avril
authors.affiliation: Division of Bacteriology, National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Institut de Recherche Biomédicale des Armées (IRBA-CRSSA), Département de Microbiologie, Unité de biotechnologie des anticorps et des toxines, 24 avenue des Maquis du Grésivaudan, B.P. 87, 38702 La Tronche Cedex, France.
authors.forename: Siham
authors.initials: S
authors.lastname: Chahboun
authors.affiliation: University of Helsinki, Faculty of Veterinary Medicine, Centre of Excellence in Microbial Food Safety Research, Department of Food Hygiene and Environmental Health, P.O. Box 66 (Agnes Sjöbergin katu 2), 00014 Helsinki University, Helsinki, Finland.
authors.forename: Hannu
authors.initials: H
authors.lastname: Korkeala
authors.affiliation: Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 25 avenue du Docteur Roux, 75015, Paris, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Mazuet
authors.affiliation: Unité des Bactéries anaérobies et Toxines, Institut Pasteur, 25 avenue du Docteur Roux, 75015, Paris, France.
authors.forename: Michel-Robert
authors.initials: MR
authors.lastname: Popoff
authors.affiliation: Institut de Recherche Biomédicale des Armées (IRBA-CRSSA), Département de Microbiologie, Unité de biotechnologie des anticorps et des toxines, 24 avenue des Maquis du Grésivaudan, B.P. 87, 38702 La Tronche Cedex, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Pelat
authors.affiliation: Institut de Recherche Biomédicale des Armées (IRBA-CRSSA), Département de Microbiologie, Unité de biotechnologie des anticorps et des toxines, 24 avenue des Maquis du Grésivaudan, B.P. 87, 38702 La Tronche Cedex, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Thullier
authors.affiliation: Division of Bacteriology, National Institute for Biological Standards and Control (NIBSC), a centre of Medicines and Healthcare products Regulatory Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Sesardic
authors.affiliation: Technische Universität Braunschweig, Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung Biotechnologie, Spielmannstr. 7, 38106 Braunschweig, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000627:therapeutic use*; D001905:Botulinum Toxins / Q000276:immunology*; D001906:Botulism / Q000188:drug therapy* / Q000276:immunology; D003014:Clostridium botulinum; D018604:Epitope Mapping; D006801:Humans; D008251:Macaca; D051379:Mice; D057127:Single-Chain Antibodies
chemical_list: D057134:Antibodies, Neutralizing; D057127:Single-Chain Antibodies; D001905:Botulinum Toxins; C096326:botulinum toxin type E