Generation of therapeutic antibodies to serotype F botulism

描述(申请人提供)：该项目的目标是开发一种高度有效的抗毒素，用于治疗和预防由F型肉毒神经毒素(BONT/F)引起的肉毒杆菌中毒。抗毒素将由三个或四个人类或人源化的单抗(MAbb)组成，这些单抗与七个BoNT/F亚型结合并中和。BoNTs是生物恐怖主义的六种最高风险威胁因子之一，因为它们具有极强的效力和致命性，易于生产，需要长期的重症监护。治疗的主要药物是马的抗毒素，这是由高度免疫的马制造的。马抗毒素对亚血清型的效力降低，过敏反应发生率显著，血清半衰期短，导致再中毒，给药具有挑战性，不能预防性给药。我们已经产生了三种单抗的组合，它们结合了所有测试的BoNT/A、B或E亚血清型，并在体内导致了高度有效的BoNT中和。结合三个结合非重叠表位的单抗，由于快速从循环中清除BONT，从而导致高度有效的BONT中和。基于这种效力，HHS已将合同授予加利福尼亚州伯克利的XOMA(US)LLC，以开发这些临床使用的mAb组合。BONT/A单抗组合已经完成了第一阶段临床试验的剂量。BONT/B和BONT/E单抗组合将于2014年第二季度进入临床试验。BONT/F特别具有挑战性，因为有7个已知的亚血清型，在氨基酸水平上彼此之间的差异高达33%。亚血清型序列变异导致马抗毒素效力降低，并使寻找结合和中和所有亚血清型的单抗具有挑战性。在NIAID U01的资助下，我们已经开发了一组结合所有或许多BoNT/F亚血清型的主要单抗。我们还表明，当这些单抗中的三个与BoNT/F亚型具有高亲和力时，就会发生有效的体内中和作用。对于这个项目，我们假设这些前导单抗可以被设计成提高与BoNT/F亚型的亲和力和交叉反应，然后发展成三到四个mAb组合，有效地中和所有BoNT/F亚型。我们将通过完成以下具体目标来实现这一目标。目的1.评估和优化单个铅单抗的特性，以确定结合和中和已报道的7个BONT/F亚型的三到四个单抗的铅组合。目的2.产生无毒的BONT/F结构域，并针对每个单抗进行特异性检测。目的3.建立稳定表达单抗的细胞系。目的4.用两步层析法进行初步工艺考察，获得纯度为90%的抗体。在这个项目完成后，我们将有一个临床前的主要候选BONT/F抗毒素，由三到四个具有可接受的人体组织交叉反应的人或人源化单抗组成，结合和中和所有七个BONT/F亚型，并拥有稳定的表达这些抗体的细胞系。本项目所获得的稳定的CHO细胞系可以作为GMP生产和临床开发的主细胞库和工作细胞库在未来快速发展。

DESCRIPTION (provided by applicant): The goal of this project is to develop a highly potent antitoxin for the treatment and prevention of botulism caused by type F botulinum neurotoxin (BoNT/F). The antitoxin will consist of a combination of three or four human or humanized monoclonal antibodies (mAbs) which together bind to and neutralize the seven BoNT/F sub-serotypes. BoNTs are one of the six highest-risk threat agents for bioterrorism, due to their extreme potency and lethality, ease of production, and need for prolonged intensive care. The mainstay of treatment is equine antitoxin, made by hyperimmunizing horses. Equine antitoxin has a reduced potency for sub-serotypes, a significant incidence of allergic reactions, a short serum half-life leading to reintoxication, is challenging to administer, and cannot be given prophylactically. We have generated combinations of three mAbs that bind all sub-serotypes tested of BoNT/A, B, or E and result in highly potent BoNT neutralization in vivo. Combining three mAbs binding non-overlapping epitopes leads to highly potent BoNT neutralization due to rapid clearance of BoNT from the circulation. Based on the potency, HHS has awarded contracts to XOMA (US) LLC, Berkeley, CA to develop these mAb combinations for clinical use. The BoNT/A mAb combination has completed dosing in a Phase 1 clinical trial. The BoNT/B and BoNT/E mAb combinations will enter clinical trials by second quarter 2014. BoNT/F is particularly challenging, as there are seven known sub-serotypes, which differ from each other by up to 33% at the amino acid level. Sub-serotype sequence variation results in reduced potency of equine antitoxin and makes finding mAbs that bind and neutralize all sub-serotypes challenging. Under NIAID U01 funding, we have developed a panel of lead mAbs binding all or many of the BoNT/F sub- serotypes. We have also shown that when three of these mAbs bind with high affinity to a BoNT/F sub- serotype, potent in vivo neutralization occurs. For this projec, we hypothesize that these lead mAbs can be engineered to improve affinity and cross reactivity for the BoNT/F sub-serotypes and then developed into a three or four mAb combination that potently neutralizes all BoNT/F sub-serotypes. We will accomplish this by completing the following specific aims. Aim 1. Evaluate and optimize individual lead mAb characteristics to identify lead combinations of three or four mAbs that bind and neutralize the seven reported BoNT/F sub-serotypes. Aim 2. Generate non-toxic BoNT/F domains and assays specific for each individual mAb. Aim 3. Develop stable cell lines that express mAbs. Aim 4. Conduct initial process investigations using two-step chromatography and achieve 90% pure antibody. At the completion of this project, we will have a preclinical lead candidate BoNT/F antitoxin consisting of three or four human or humanized mAbs that have acceptable human tissue cross reactivity, bind and neutralize all seven BoNT/F sub-serotypes, and have stable cell lines that express these antibodies. The stable CHO cell lines produced in this project can be rapidly developed in the future as master and working cell banks for GMP manufacture and clinical development.