Next Generation pan-serotype,antitoxin for botulinum neurotoxin exposures

About This Grant

Botulinum neurotoxins (BoNTs) inactivate motor neurons with extraordinary potency causing flaccid paralysis, breathing difficulties and often death. As such, BoNTs are among the most dangerous Tier 1 biothreat agents, and in addition, cause significant death and illness from botulism poisoning obtained from contaminated foods. The few available botulism treatments derive from polyclonal antisera produced in large animals, such as the equine botulism antitoxin ‘BAT’. Such products are often associated with multiple commercial challenges and potential health risks. The BoNT antitoxins are centrally stockpiled which often causes significant delays in the time to treatment, thus reducing or even obviating their efficacy as they must be administered before the circulating toxin has entered neurons. Our strategy for a next generation BoNT antitoxin product is to employ heteromultimers of linked antitoxin camelid single-domain antibodies (VHHs or nanobodies), likely administered encoded as formulated RNA. Our RNA products will permit economic manufacturing and distributed stockpiling, thus reducing time to treatment and improving efficacy. We have previously shown that heteromultimeric VHH- based neutralizing agents (VNAs) containing six VHH components can have high potencies equivalent to or greater than those of complex polyclonal antisera or conventional monoclonal antibody antitoxin products. Furthermore, VNA products offer exceptional versatility in product design. Formulated RNA products have been amply demonstrated as safe, rapidly responsiveness to new threats and rapidly and economically manufactured at large scale. In our studies, we have shown that a single protein VNA or a formulated RNA/VNA product can provide potent antitoxin protection from at least three different botulism serotypes in toxin exposed animals. In the R61 phase of this R61/R33 proposal, we will use our robust computational VHH discovery tools together with detailed characterization of binding structures to complete the discovery of VHH components that broadly bind and neutralize all known natural subtypes of the seven BoNT serotypes. In the R33 phase, we will create one or two VHH heteromultimeric VNAs and test their ability to provide potent protection from all natural subtypes of the seven BoNT serotypes. We will perform extensive process and product development activities for a VNA Drug Product and perform IND-enabling studies, including testing efficacy in mouse and guinea pig models of BoNT intoxication. If successfully completed, we will have demonstrated the feasibility of a next-generation antitoxin product to replace the current, outdated, polyclonal BoNT antitoxin and be in position to rapidly commercialize the new product. Also important, the technologies we develop should have broad application in creating practical and effective antitoxin therapeutics for many currently untreated or ineffectively treated toxin- mediated diseases.