Trispecific monoclonal antibody for botulinum neurotoxin intoxication therapy

描述（由申请方提供）：本项目的目标是产生用于预防和治疗肉毒神经毒素（BoNT）血清型A、B、E和F引起的肉毒中毒的高效三特异性抗体。由于其极强的效力和致命性、易于生产以及需要长期重症监护，BoNT是生物恐怖主义六种风险最高的威胁因子之一。马抗毒素过敏反应的发生率高，血清半衰期短，导致再中毒，给药具有挑战性，且不能一次性给药。考虑到BoNTs的特殊毒性，抗毒素必须具有非常高的效力。先前的研究已经发现，没有单一mAb以所需的效力中和BoNT，然而，由于BoNT从循环中的快速清除，结合非重叠表位的mAb导致高度有效的BoNT中和。我们已经产生了结合BoNT/A、B或E的所有亚型的三种mAb的组合，并导致体内高度有效的BoNT中和。根据效力，HHS已与一家生物技术公司（XOMA（US）LLC）签订合同，开发这些mAb组合用于临床。BoNT/A三mAb组合已进入1期临床试验。BoNT/B和BoNT/E三种mAb组合正在cGMP生产中，并将于2013-2014年进入临床试验。这种方法的挑战是需要生产和质量控制大量mAb（每种血清型3种）。这增加了产品的成本和复杂性，特别是如果将所有mAb组合以创建多血清型药物。我们提出的替代方案是为每种血清型创建单个三特异性mAb，并结合三种mAb中每种的结合位点。对于这项工作，我们假设可以产生这样的三特异性mAb，其将具有与三种mAb组合相同的结合和BoNT中和特性。在R21部分中，我们将构建结合三个非重叠BoNT/A表位的三特异性抗体（TsAb）。将构建四种不同的TsAb，其在三个可变结构域中的每一个相对于抗体Fc的位置方面不同。每种TsAb将在对三个表位中的每一个的亲和力、与BoNT/A结合的化学计量、稳定性以及表达和纯化的容易性方面进行表征和优化。将在小鼠中和测定中评价多种TsAb构建体的体内药代动力学（PK）和BoNT/A清除和中和效力。体内研究将由我们的长期合作者Luisa Cheng博士在USDA进行。这些研究将鉴定具有最佳性质的那些构建体，并将产生有效中和BoNT/A的单一mAb。在该授权的R33部分，BoNT/A鉴定的最佳构建体将应用于其他三种BoNT血清型，B、E和F。该项目的结果将是抗体，将治疗超过99%的人类肉毒杆菌中毒病例。 公共卫生相关性：该项目的目标是产生高效和安全的抗体，用于预防和治疗肉毒杆菌神经毒素血清型A、B、E和F引起的肉毒杆菌中毒。BoNT是生物恐怖主义的六种最高风险威胁因子之一，目前FDA批准的马抗毒素不适用于大规模暴露事件。该项目将产生具有成本效益的重组抗体，用于治疗99%的人类肉毒杆菌中毒病例，并准备进行安全测试。

DESCRIPTION (provided by applicant): The goal of this project is to generate highly potent trispecific antibodies for the prevention and treatment of botulism due to botulinum neurotoxins (BoNT) serotypes A, B, E, and F. 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. Equine antitoxin has a high incidence of allergic reactions, a short serum half-life leading to reintoxication, is challenging to administer, and cannot be given prophylactically. Given the extraordinary toxicity of BoNTs, antitoxin must be of very high potency. Previous studies have found that no single mAb neutralizes BoNT with the requisite potency, however, combining mAbs binding non-overlapping epitopes leads to highly potent BoNT neutralization due to rapid clearance of BoNT from the circulation. We have generated combinations of three mAbs that bind all subtypes of BoNT/A, B, or E and result in highly potent BoNT neutralization in vivo. Based on the potency, HHS has awarded contracts to a biotechnology company (XOMA (US) LLC) to develop these mAb combinations for clinical use. The BoNT/A three mAb combination has entered phase 1 clinical trials. The BoNT/B and BoNT/E three mAb combinations are in cGMP manufacturing and will enter clinical trials by 2013-2014. A challenge of this approach is the large number of mAbs that need to be manufactured and quality controlled (3 per serotype). This adds to the cost and complexity of the product, especially if the mAbs are all going to be combined to create a multi-serotype drug. Our proposed alternative is to create a single trispecific mAb for each serotype incorporating the binding sites of each of the three mAbs. For this work, we hypothesize that such trispecific mAb can be generated which will have the same binding and BoNT neutralization properties as the three mAb combination. In the R21 portion we will construct trispecific antibodies (TsAbs) binding three non-overlapping BoNT/A epitopes. Four different TsAbs will be constructed that differ with respect to where each of the three variable domains are positioned relative to the antibody Fc. Each TsAb will be characterized and optimized with respect to affinity for each of the three epitopes, stoichiometry of binding to BoNT/A, stability, and ease of expression and purification. Multiple TsAb constructs will be evaluated for in vivo pharmacokinetics (PK) and BoNT/A clearance and neutralization potency in the mouse neutralization assay. In vivo studies will be conducted at the USDA by our long-term collaborator, Dr. Luisa Cheng. These studies will identify those constructs with optimal properties and will result in a single mAb that potentl neutralizes BoNT/A. In the R33 portion of this grant, the optimal construct identified for BoNT/A will be applied to three other BoNT serotypes, B, E and F. The result of this project will be antibodies that would treat over 99% of human botulism cases. PUBLIC HEALTH RELEVANCE: The goal of this project is to generate highly potent and safe antibodies for the prevention and treatment of botulism due to botulinum neurotoxins serotypes A, B, E, and F. BoNTs are one of the six highest-risk threat agents for bioterrorism, and current FDA-approved equine antitoxin is untenable for use in mass exposure events. This project will generate cost-effective recombinant antibodies to treat the cause of 99% of the cases of human botulism that will be ready for safety testing.