Consuelo Garcia-Rodriguez

Recombinant monoclonal antibody (mAb) botulinum neurotoxin (BoNT) antitoxins, consisting of three mAbs that bind non-overlapping epitopes, are highly potent. However, the three-mAb mixtures pose unique development and manufacturing challenges. Combining even more mAbs to create multivalent antitoxin drugs multiplies those challenges. We previously reported that a single tri-epitopic IgG1-based mAb (TeAb) containing the variable domains of the three parental BoNT/A mAbs and an Fc was as potent as the combination of three IgGs in the mouse neutralization assay (MNA). Here, we extended the tri-epitopic strategy to three other BoNT serotypes. Each TeAb (TeAb-B for BoNT/B, TeAb-E for BoNT/E, and TeAb-F for BoNT/F) binding was measured using fluorescence-activated cell sorting and flow fluorimetry, and the potency was tested in the MNA. The three TeAbs displayed binding affinities that were the same within error of the parental IgGs for each epitope, and all had higher avidity to each serotype of BoNT than that of the parental mAbs. The potency of the BoNT/B, BoNT/E, and BoNT/F TeAbs was similar to the combinations of the three parental IgGs binding BoNT/B, BoNT/E, and BoNT/F in the MNA. We now have four examples of a single TeAb recapitulating the affinity and in vivo potency of a three-mAb antitoxin. The tri-epitopic strategy could be applied to streamline the production and bioanalytics of antibody drugs where three-mAb binding is required for activity.

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 K_D values ranging from 3.86 nM to 103 nM (median K_D 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 K_D 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.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.