A Three-Monoclonal Antibody Combination Potently Neutralizes BoNT/G Toxin in Mice

Toxins (Basel). 2023 Apr 30;15(5):316. doi: 10.3390/toxins15050316.

Abstract

Equine-derived antitoxin (BAT®) is the only treatment for botulism from botulinum neurotoxin serotype G (BoNT/G). BAT® is a foreign protein with potentially severe adverse effects and is not renewable. To develop a safe, more potent, and renewable antitoxin, humanized monoclonal antibodies (mAbs) were generated. Yeast displayed single chain Fv (scFv) libraries were prepared from mice immunized with BoNT/G and BoNT/G domains and screened with BoNT/G using fluorescence-activated cell sorting (FACS). Fourteen scFv-binding BoNT/G were isolated with KD values ranging from 3.86 nM to 103 nM (median KD 20.9 nM). Five mAb-binding non-overlapping epitopes were humanized and affinity matured to create antibodies hu6G6.2, hu6G7.2, hu6G9.1, hu6G10, and hu6G11.2, with IgG KD values ranging from 51 pM to 8 pM. Three IgG combinations completely protected mice challenged with 10,000 LD50s of BoNT/G at a total mAb dose of 6.25 μg per mouse. The mAb combinations have the potential for use in the diagnosis and treatment of botulism due to serotype G and, along with antibody combinations to BoNT/A, B, C, D, E, and F, provide the basis for a fully recombinant heptavalent botulinum antitoxin to replace the legacy equine product.

Keywords: antibody engineering; botulinum neurotoxin; mouse neutralization assay; oligoclonal antibodies; recombinant antibodies; serotype G botulism.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal
  • Antitoxins*
  • Botulinum Toxins, Type A*
  • Botulism* / prevention & control
  • Horses
  • Immunoglobulin G
  • Mice
  • Saccharomyces cerevisiae / metabolism
  • Single-Chain Antibodies*

Substances

  • Antibodies, Monoclonal
  • Botulinum Toxins, Type A
  • Single-Chain Antibodies
  • Antitoxins
  • Immunoglobulin G

Grants and funding

This research was funded by the National Institute of Allergy and Infectious Diseases (NIAID), under contract number HHS75N93019C00057 to Ology Bioservices Inc. L.W.C., C.C.T. and P.A. were funded by the United States Department of Agriculture, Agriculture Research Service, National Program 108, CRIS project 2030-42000-053-00D.