A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes

Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.

人类肉毒中毒主要由肉毒神经毒素（botulinum neurotoxin，BoNT）血清型A、B及E型引发，约1%的病例由F型肉毒神经毒素（BoNT/F）导致。BoNT/F至少包含7种不同亚型，各亚型间的氨基酸序列差异最高可达36%。此类序列差异为开发可结合、检测并中和所有BoNT/F亚型的单克隆抗体（monoclonal antibodies，mAbs）带来了显著挑战。我们通过免疫小鼠抗体可变区（V区）库克隆与酵母展示技术，成功获得33株先导单链可变区片段（single chain Fv，scFv）单克隆抗体组合，这些抗体可结合一种或多种BoNT/F亚型，其中位平衡解离常数（equilibrium dissociation constant，KD）为4.06×10⁻⁹ M。通过对先导单克隆抗体的可变区进行序列多样化改造并筛选具有交叉反应性的结合物，我们最终得到5株可结合全部7种BoNT/F亚型的单克隆抗体。将其中结合非重叠表位的3株scFv转化为免疫球蛋白G（IgG）后，其针对不同BoNT/F亚型的KD值范围为2.2×10⁻⁸ M至1.47×10⁻¹² pM。在小鼠中和实验中，等摩尔比例混合的该系列单克隆抗体可有效中和BoNT/F1、F2、F4及F7型毒素。该系列单克隆抗体具备开发为可识别所有已知BoNT/F亚型的诊断试剂的潜力，亦可被开发为抗毒素，用于预防和治疗F型肉毒中毒。