A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes

Human botulismis most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxincapable of binding and neutralizing multiple subtypesof BoNT/E. Libraries of yeast-displayedsingle chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent- toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting(FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypeswith nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypestested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypesranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical developmentwith an investigational new drug(IND) application filing expected in 2018.