A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D

Abstract

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.

Multicolor fluorescence activated cell sorting to generate humanized monoclonal antibody binding seven subtypes of BoNT/F

“…High affinity is crucial for neutralizing mAbs to clear and neutralize botulinum neurotoxins (BoNT) because of their extreme potency [ 40 ]. It has been established that a combination of at least three mAbs with high affinity is required to neutralize BoNT with a potency that allows a clinically reasonable dose, while any single mAb even with extremely high affinity (K D = 56 pM) does not achieve this potency [ 13 , 16 – 18 ]. In addition, cross-specificity of mAbs is necessary since each BoNT serotype usually contains multiple subtypes.…”

“…As an alternative to equine derived antitoxin [15], highly potent human mAb-based antitoxins composed of three mAbs [13,[16][17][18] are being developed for serotypes A, B, C, D, E, F, and G with the most advanced (serotypes A, B, C, D, and E) having completed Phase 1 human testing [19][20][21][22]. The three mAbs bind non-overlapping epitopes on the BoNT molecule and elicit rapid clearance from the circulation and high potency [23,24].…”

Toxicology and pharmacology of botulinum and tetanus neurotoxins: an update

“…This orients the BoNT molecule while approaching the plasma membrane thus favoring their landing with the HC-C domain and increasing the probability of membrane binding (Fogolari et al 2009 ). The role of PSGs as primary receptors is supported by several experimental evidence (Binz and Rummel 2009 ), and by the fact that the most TeNT- or BoNT-neutralizing monoclonal antibodies sterically prevent PSG binding (Garcia-Rodriguez et al 2007 , 2021 ; Mukherjee et al 2012 ; Lam et al 2018 ; Tomic et al 2021 ; Pirazzini et al 2021 ; Wang et al 2016 , 2021 ; Zhang et al 2021 ; Brier et al 2021 ).…”

“…Accordingly, therapy by intratechal administration of antibodies is more efficacious than intravenous infusion because the TeNT binding antibodies are infused in the anatomical site of action of this neurotoxin (Kabura et al 2006 ). Research on anti-TeNT and anti-BoNTs antibodies has been discussed in previous reviews and papers (Chen et al 2012 ; Mukherjee et al 2012 ; Rasetti- Escargueil and Popoff 2019 ; Garcia-Rodriguez et al 2007 , 2021 ; Lam et al 2020 ; Snow et al 2019 ; Brier et al 2021 ; Tomic et al 2021 ), and we refer to them and to references cited therein, whilst only more recent reports are discussed here.…”

“…HumAbs as well as camelid single domain antibodies specific for different BoNT serotypes have been prepared and shown to prevent botulism when injected before or together with BoNTs. In addition, it appears that BoNTs are less immunogenic that TeNT and that combination of three/four antibodies are to be used to neutralize completely one BoNT serotype (Chen et al 2012 ; Mukherjee et al 2012 ; Garcia-Rodriguez et al 2007 , 2021 ; Lam et al 2018 ; Snow et al 2019 ; Tomic et al 2021 ; and refences contained therein).…”