Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

MAbs. Mar-Apr 2014;6(2):446-59. doi: 10.4161/mabs.27773. Epub 2014 Jan 9.

Abstract

Botulinum toxins (BoNTs) are among the most toxic substances on earth, with serotype A toxin being the most toxic substance known. They are responsible for human botulism, a disease characterized by flaccid muscle paralysis that occurs naturally through food poisoning or the colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNT has been classified as a category A agent by the Centers for Disease Control, and it is one of six agents with the highest potential risk of use as bioweapons. Human or human-like neutralizing antibodies are thus required for the development of anti-botulinum toxin drugs to deal with this possibility. In this study, Macaca fascicularis was hyperimmunized with a recombinant light chain of BoNT/A. An immune phage display library was constructed and, after multistep panning, several scFv with nanomolar affinities that inhibited the endopeptidase activity of BoNT/A1 in vitro as scFv-Fc, with a molar ratio (ab binding site:toxin) of up to 1:1, were isolated. The neutralization of BoNT/A-induced paralysis by the SEM120-IID5, SEM120-IIIC1 and SEM120-IIIC4 antibodies was demonstrated in mouse phrenic nerve-hemidiaphragm preparations with the holotoxin. The neutralization observed is the strongest ever measured in the phrenic nerve-hemidiaphragm assay for BoNT/A1 for a monoclonal antibody. Several scFv-Fc inhibiting the endopeptidase activity of botulinum neurotoxin A were isolated. For SEM120-IID5, SEM120-IIIC1, and SEM120-IIIC4, inhibitory effects in vitro and protection against the toxin ex vivo were observed. The human-like nature of these antibodies makes them promising lead candidates for further development of immunotherapeutics for this disease.

Keywords: BoNT/A; Clostridium botulinum; Macaca fascicularis; SNAP-25; botulinum neurotoxin; ex-vivo assay; in vitro assay; macaque; mouse phrenic nerve-hemidiaphragm assay; phage display; scFv; scFv-Fc.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Blocking / administration & dosage
  • Antibodies, Blocking / genetics
  • Antibodies, Blocking / metabolism*
  • Botulinum Toxins, Type A / adverse effects
  • Botulinum Toxins, Type A / immunology*
  • Botulism / complications
  • Botulism / immunology
  • Botulism / therapy*
  • Cell Surface Display Techniques
  • Clostridium botulinum type A / immunology*
  • Epitope Mapping
  • Humans
  • Immunity / genetics
  • Immunization
  • Immunoglobulin Fc Fragments / genetics
  • Immunoglobulin Fc Fragments / metabolism*
  • Immunoglobulin Light Chains, Surrogate / administration & dosage
  • Immunoglobulin Light Chains, Surrogate / genetics
  • Immunoglobulin Light Chains, Surrogate / metabolism*
  • Immunotherapy / methods*
  • Macaca fascicularis
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Paralysis / etiology
  • Paralysis / immunology
  • Paralysis / prevention & control*
  • Phrenic Nerve / drug effects*
  • Phrenic Nerve / immunology
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / metabolism*

Substances

  • Antibodies, Blocking
  • Immunoglobulin Fc Fragments
  • Immunoglobulin Light Chains, Surrogate
  • Single-Chain Antibodies
  • Botulinum Toxins, Type A