Lou Jianlong, Wen Wei Hua, Conrad Fraser, Tam Christina C, Garcia-Rodriguez Consuelo, Farr-Jones Shauna, Marks James D
Department of Anesthesia and Perioperative Care, University of California, Zuckerberg San Francisco General Hospital, Box 1327, 2540 23rd St, San Francisco, CA 94143, USA.
Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Services, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA.
Toxins (Basel). 2025 Jun 4;17(6):281. doi: 10.3390/toxins17060281.
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 the 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 similar to the combinations of the three parental IgGs binding BoNT/B, BoNT/E, and BoNT/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.
重组单克隆抗体（mAb）肉毒杆菌神经毒素（BoNT）抗毒素由三种结合非重叠表位的单克隆抗体组成，效力很强。然而，三种单克隆抗体的混合物带来了独特的研发和生产挑战。组合更多单克隆抗体制备多价抗毒素药物会使这些挑战成倍增加。我们之前报道过，一种基于IgG1的单克隆三表位抗体（TeAb），包含三种亲本BoNT/A单克隆抗体的可变区和一个Fc段，在小鼠中和试验（MNA）中与三种IgG组合的效力相当。在此，我们将三表位策略扩展到其他三种BoNT血清型。使用荧光激活细胞分选和流式荧光测定法测量每种TeAb（针对BoNT/B的TeAb-B、针对BoNT/E的TeAb-E和针对BoNT/F的TeAb-F）的结合情况，并在MNA中测试其效力。这三种TeAb对每个表位的结合亲和力在亲本IgG的误差范围内相同，并且对每种BoNT血清型的亲和力都高于亲本单克隆抗体。BoNT/B、BoNT/E和BoNT/F的TeAb在MNA中的效力与结合BoNT/B、BoNT/E和BoNT/F的三种亲本IgG组合相似。我们现在有四个单克隆三表位抗体的实例，其重现了三单克隆抗毒素的亲和力和体内效力。三表位策略可应用于简化抗体药物的生产和生物分析，这类抗体药物需要三单克隆抗体结合才能发挥活性。
