Generation of therapeutic antibodies to serotype F botulism

描述（由申请人提供）：该项目的目标是开发一种高效抗毒素，用于治疗和预防由F型肉毒杆菌神经毒素（BoNT/F）引起的肉毒中毒。抗毒素将由三到四种人或人源化单克隆抗体（mab）组成，它们结合并中和七种BoNT/F亚血清型。bont是生物恐怖主义的六种最高风险威胁剂之一，因为它们具有极强的效力和杀伤力，易于生产，并且需要长期的重症监护。主要的治疗方法是马抗毒素，由高度免疫的马产生。马抗毒素对亚血清型的效力较低，过敏反应发生率高，血清半衰期短，导致再中毒，施用具有挑战性，不能预防性给予。我们已经生成了三种单抗的组合，可以结合BoNT/A、B或E测试的所有亚血清型，并在体内产生高效的BoNT中和。结合三个单抗结合非重叠表位，由于BoNT从循环中快速清除，导致高效的BoNT中和。基于效力，HHS已与XOMA (US) LLC, Berkeley， CA签订合同，开发这些单抗组合用于临床。BoNT/A mAb组合已经完成了1期临床试验的给药。BoNT/B和BoNT/E单抗组合将于2014年第二季度进入临床试验阶段。BoNT/F尤其具有挑战性，因为已知有7种亚血清型，在氨基酸水平上彼此差异高达33%。亚血清型序列变异导致马抗毒素效力降低，使得寻找结合和中和所有亚血清型的单克隆抗体具有挑战性。在NIAID U01资助下，我们开发了一组结合所有或许多BoNT/F亚血清型的先导单克隆抗体。我们还表明，当其中三种单克隆抗体与BoNT/F亚型高亲和力结合时，体内会发生有效的中和。在这个项目中，我们假设这些先导单抗可以被设计成提高BoNT/F亚血清型的亲和力和交叉反应性，然后发展成三或四单抗组合，有效中和所有BoNT/F亚血清型。我们将通过完成以下具体目标来实现这一目标。目的1。评估和优化单个单克隆抗体的特征，以确定3个或4个单克隆抗体结合并中和7个报告的BoNT/F亚血清型的先导组合。目标2。生成无毒BoNT/F结构域，并对每个单抗进行特异性检测。目标3。培养表达单克隆抗体的稳定细胞系。目标4。采用两步色谱法进行初步工艺调查，抗体纯度达到90%。在该项目完成后，我们将获得临床前主要候选BoNT/F抗毒素，该抗毒素由三到四种人或人源化单克隆抗体组成，具有可接受的人体组织交叉反应性，结合并中和所有七种BoNT/F亚血清型，并具有表达这些抗体的稳定细胞系。本项目生产的稳定的CHO细胞系可作为GMP生产和临床开发的主细胞库和工作细胞库，在未来得到快速开发。

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.