Single tri-epitopic antibodies (TeAbs) to botulinum neurotoxin serotypes B, E, and F recapitulate the full potency of a combination of three monoclonal antibodies in toxin neutralization

Authors: LOU, JIANLONG - University Of California San Francisco (UCSF); WEN, WEIHUA - University Of California San Francisco (UCSF); CONRAD, FRASER - University Of California San Francisco (UCSF); Tam, Christina; GARCIA-RODRIGUEZ, CONSUELO - University Of California San Francisco (UCSF); FARR-JONES, SHAUNA - University Of California San Francisco (UCSF); MARKS, JAMES - University Of California San Francisco (UCSF)

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/26/2025
Publication Date: 6/4/2025
Citation: Lou, J., Wen, W., Conrad, F., Tam, C.C., Garcia-Rodriguez, C., Farr-Jones, S., Marks, J.D. 2025. Single tri-epitopic antibodies (TeAbs) to botulinum neurotoxin serotypes B, E, and F recapitulate the full potency of a combination of three monoclonal antibodies in toxin neutralization. Toxins. 17(6). Article 281. https://doi.org/10.3390/toxins17060281.
DOI: https://doi.org/10.3390/toxins17060281

Interpretive Summary: Botulinum neurotoxin (BoNT) is one of the most toxic substances known, classified as a Select Agent, and capable of being used as a bio-weapon. It has been shown that recombinant monoclonal antibody antitoxins consisting of three mAbs directed to each BoNT serotype that bind non-overlapping epitopes are highly potent but their manufacturing and production are challenging. We have shown that a single tri-epitopic IgG1-based mAb (TeAB) can recapitulate the antibody protection of using all three mAbs in the mouse neutralization assay. In this study, we show that we can use the same strategy to develop three other TeAbs against BoNT/B, BoNT/E, and BoNT/F that were protective against BoNT intoxication. These results indicate that this single-epitopic strategy is potent and can be applied for the production of other antibody-based products that need multiple antibodies for neutralization.

Technical Abstract: 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 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 the same within error of the combinations of the three parental IgGs binding BoNT/B E and 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.