Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B

PLoS One. 2016 Aug 25;11(8):e0161446. doi: 10.1371/journal.pone.0161446. eCollection 2016.

Abstract

Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known 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. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development.

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Humanized / immunology*
  • Antibodies, Neutralizing / immunology
  • Botulinum Toxins, Type A / immunology*
  • Botulism / immunology
  • Clostridium botulinum
  • Female
  • Humans
  • Immunoglobulin G / immunology
  • Macaca fascicularis / immunology
  • Mice
  • Neutralization Tests
  • Single-Chain Antibodies / immunology

Substances

  • Antibodies, Monoclonal, Humanized
  • Antibodies, Neutralizing
  • Immunoglobulin G
  • Single-Chain Antibodies
  • rimabotulinumtoxinB
  • Botulinum Toxins, Type A

Grant support

We acknowledge funding from the European Community’s Seventh Framework Program (FP7/2007-2013) under agreement no. 241832 granted to the AntiBotABE project. LFB Biotechnologies provided support in the form of salaries for authors RU and A. Fontayne, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.