A three-monoclonal antibody combination potently neutralizes BoNT/G toxin in mice

Authors: FAN, YONGFENG - University Of California San Francisco (UCSF); LOU, JIANLONG - University Of California San Francisco (UCSF); Tam, Christina; WEN, WEIHUA - University Of California San Francisco (UCSF); CONRAD, FRASER - University Of California San Francisco (UCSF); ALVES, PRISCILA - Orise Fellow; CHENG, LUISA - Former ARS Employee; GARCIA-RODRIGUEZ, C - 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: 2/25/2023
Publication Date: 4/30/2023
Citation: Fan, Y., Lou, J., Tam, C.C., Wen, W., Conrad, F., Alves, P., Cheng, L.W., Garcia-Rodriguez, C., Farr-Jones, S., Marks, J.D. 2023. A three-monoclonal antibody combination potently neutralizes BoNT/G toxin in mice. Toxins. 15(5). Article 316. https://doi.org/10.3390/toxins15050316.
DOI: https://doi.org/10.3390/toxins15050316

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. At least seven serotypes of BoNT/A-G are well documented and new BoNT variants or BoNT-like proteins have been reported including BoNT/H (or HA), BoNT/X, eBoNT/J, and BoNT/Wo. Most naturally occurring human botulism is caused by BoNT serotypes A, B, E and F, and is rarely associated with BoNT/G. However, BoNT/G has been suspected as the cause of deaths in Switzerland thus confirming the human lethality of this serotype. Currently, the only available countermeasure and treatment to botulism in adults is the equine-derived heptavalent botulinum antitoxin (BAT®). However, this treatment in humans can cause undesired side-effects along with reduced effectiveness and relapse of botulism after treatment. Thus new therapeutics targeting all seven BoNT serotypes as a heptavalent antitoxin are critically needed.

Technical Abstract: Botulinum neurotoxin serotype G (BoNT/G) is produced by C. argentinense as a single-chain protein, that shares the most similarity to the BoNT/B Okra (58.2%) and the least to BoNT/C Stockholm (32.9%). Equine-derived antitoxin or BAT® is the only treatment for BoNT/G intoxication but with low efficacy and potentially severe adverse effects. Yeast-displayed BoNT/G LCHN, G LCHN plus crude G toxin, or recombinant catalytically inactive BoNT/G (Gi) -immunized scFv libraries were constructed and screened with BoNT/Gi using fluorescence-activated cell sorting (FACS). The individual colonies were sequenced, and KD values were measured. Recombinant BoNT/G domains LC, LCHN, HC were prepared in E. coli. Over sixteen mAbs against BoNT/Gi were isolated from the libraries by sorting with BoNT/Gi. The KD value on the yeast surface was measured ranging from 3.86 nM to 148 nM, with a median KD value of 20.9 nM. Among the mAbs, two mAbs did not bind active BoNT/G holotoxin or any of the BoNT/G domains, two mAbs bound BoNT/G LC, three bound BoNT/G LCHN, and nine bound on BoNT/G Hc. Competition analysis with flow cytometry demonstrated that the sixteen mAbs bound on eight different epitopes on BoNT/Gi, two on LC, two on LCHN, and four on HC. Five non-overlapping mAbs were humanized and affinity matured to hu6G6.1, hu6G7.2, hu6G9.1, hu6G10, and hu6G11.2, the KD values measured by KinExA ranged from 51 to 8 pM. Any three-mAb combination of any of the five mAbs at a dose of 12.5'g per mouse neutralized 10,000 MLD50s of BoNT/G. Thus, we identified multiple three-mAb combinations with high affinity to BoNT/G that potently neutralize BoNT/G in the mouse neutralization assay (MNA). The mAbs have the potential for use in diagnosis and treatment of botulism due to serotype G and are the final mAbs required to formulate a fully recombinant heptavalent botulinum antitoxin to replace the legacy equine product.