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.

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

“…32 and 6). We have found that VHH heteromultimer VNAs possess additional potential therapeutic advantages over conventional mAb products such as 1) the option to co-administer an anti-tag effector antibody that binds multiple tags on the VNAs (11,13) to promote antibody Fc effector functions such as serum clearance (33), 2) the ability to target multiple toxins with one biomolecule (12,16), and 3) the use of VNA gene therapy as an effective means to provide prolonged antitoxin protection (34,35). Simple addition of an albumin binding peptide (34,36) can dramatically improve the serum stability of VNAs (34).…”

Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies

“…One possibility for this enhanced efficacy was accelerated clearance of the toxin through the FcR pathway. The therapeutic benefit of rapid clearance has been shown for botulinum toxin (43,44) where mAbs binding to epitope tags decorate the toxin with multiple Fc domains, leading to accelerated FcR-mediated clearance. We confirmed that such a mechanism was activated when mAb 20B1 and mAb 14G8 were used in combination, and thus accelerated clearance of SEB from mouse serum via an FcR-mediated pathway was documented (Fig.…”

A Single VHH-Based Toxin-Neutralizing Agent and an Effector Antibody Protect Mice against Challenge with Shiga Toxins 1 and 2

“…To promote toxin clearance, the VNA can be coadministered with a single antitag MAb, the efAb, that binds to multiple epitopic tags engineered into each VNA molecule. When VNAs are bound at separate sites on the toxin and each VNA is bound to two or more efAbs through the tags, the toxin becomes decorated by sufficient efAbs to promote liver clearance (30), presumably by low-affinity FcRs.…”