Generation of therapeutic antibodies to serotype F botulism

描述（由申请人提供）：本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。抗毒素将由三种或四种人或人源化单克隆抗体（mAb）的组合组成，其一起结合并中和七种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致命性，易于生产，并且需要长期重症监护。治疗的主要方法是马抗毒素，由高度免疫的马制成。马抗毒素对亚血清型的效力降低，过敏反应的发生率显著，血清半衰期短，导致再中毒，给药具有挑战性，并且不能一次性给药。我们已经产生了三种mAb的组合，其结合BoNT/A、B或E的所有测试的亚血清型，并导致体内高度有效的BoNT中和。结合非重叠表位的三种mAb的组合由于BoNT从循环中的快速清除而导致高度有效的BoNT中和。根据效力，HHS已与XOMA（US）LLC（Berkeley，CA）签订合同，开发这些mAb组合用于临床。BoNT/A mAb组合已在1期临床试验中完成给药。BoNT/B和BoNT/E mAb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，因为有七种已知的血清亚型，它们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的效力降低，并使得发现结合和中和所有亚血清型的mAb具有挑战性。在NIAID U 01资助下，我们开发了一组结合所有或许多BoNT/F亚型血清型的先导mAb。我们还表明，当这些mAb中的三种以高亲和力结合BoNT/F亚血清型时，发生有效的体内中和。对于该项目，我们假设这些先导mAb可以被工程化以提高对BoNT/F亚型血清型的亲和力和交叉反应性，然后开发成有效中和所有BoNT/F亚型血清型的三种或四种mAb组合。我们将通过完成以下具体目标来实现这一目标。目标1。评估和优化单个先导mAb特征，以鉴定结合和中和7种报告的BoNT/F亚型血清型的3种或4种mAb的先导组合。目标2.生成无毒BoNT/F结构域和对每个mAb具有特异性的测定。目标3.开发表达mAb的稳定细胞系。目标4。使用两步色谱法进行初始工艺研究，并获得90%纯度的抗体。在该项目完成时，我们将获得临床前主要候选BoNT/F抗毒素，其由三种或四种人或人源化mAb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亚型血清型，并具有表达这些抗体的稳定细胞系。本项目生产的稳定CHO细胞系可在未来快速开发，作为GMP生产和临床开发的主细胞库和工作细胞库。

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.