title: Efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents.
abstract: Antitoxins for botulinum neurotoxins (BoNTs) and other toxins are needed that can be produced economically with improved safety and shelf-life properties compared to conventional therapeutics with large-animal antisera. Here we show that protection from BoNT lethality and rapid BoNT clearance through the liver can be elicited in mice by administration of a pool of epitope-tagged small protein binding agents together with a single anti-tag monoclonal antibody (MAb). The protein binding agents used in this study were single-chain Fv domains (scFvs) with high affinity for BoNT serotype A (BoNT/A). The addition of increasing numbers of differently tagged scFvs synergistically increased the level of protection against BoNT/A. It was not necessary that any of the BoNT/A binding agents possess toxin-neutralizing activity. Mice were protected from a dose equivalent to 1,000 to 10,000 50% lethal doses (LD(50)) of BoNT/A when given three or four different anti-BoNT scFvs, each fused to an E-tag peptide, and an anti-E-tag IgG1 MAb. Toxin protection was enhanced when an scFv contained two copies of the E tag. Pharmacokinetic studies demonstrated that BoNT/A was rapidly cleared from the sera of mice given a pool of anti-BoNT/A scFvs and an anti-tag MAb but not from the sera of mice given scFvs alone or anti-tag MAb alone. The scFv pool and anti-tag MAb protected mice from lethality when administered up to 2 h following exposure of mice to a dose equivalent to 10 LD(50) of BoNT/A. These results suggest that it will be possible to rapidly and economically develop and produce therapeutic antitoxins consisting of pools of tagged binding agents that are administered with a single, stockpiled anti-tag MAb.
authors.affiliation: Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, 200 Westboro Rd., North Grafton, MA 01536, USA.
authors.forename: Jorge
authors.initials: J
authors.lastname: Sepulveda
authors.forename: Jean
authors.initials: J
authors.lastname: Mukherjee
authors.forename: Saul
authors.initials: S
authors.lastname: Tzipori
authors.forename: Lance L
authors.initials: LL
authors.lastname: Simpson
authors.forename: Charles B
authors.initials: CB
authors.lastname: Shoemaker
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000494:pharmacology; D001904:Botulinum Antitoxin / Q000276:immunology* / Q000494:pharmacology; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology / Q000494:pharmacology; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000494:pharmacology; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D001904:Botulinum Antitoxin; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019274:Botulinum Toxins, Type A