Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B

作者：Rasetti Escargueil Christine; Avril Arnaud; Chahboun Siham; Tierney Rob; Bak Nicola; Miethe Sebastian; Mazuet Christelle; Popoff Michel R; Thullier Philippe; Hust Michael; Pelat Thibaut; Sesardic Dorothea^*

来源：mAbs, 2015, 7(6): 1161-1177.

DOI：10.1080/19420862.2015.1082016

• botulinum neurotoxins
• BoNT/B
• VAMP2
• in vitro endopeptidase
• ex vivo mouse phrenic nerve-hemidiaphragm
• neutralization assays
• scFv-Fc
• mouse paralysis
• synergistic protection macaque

Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which suggest that they may be well tolerated in potential clinical development.

• 出版日期2015-11-2
• 单位中国食品药品检定研究院