Trispecific monoclonal antibody for botulinum neurotoxin intoxication therapy

描述(申请人提供)：该项目的目标是产生高度有效的三特异性抗体，用于预防和治疗由A型、B型、E型和F型肉毒神经毒素(BoNT)引起的肉毒杆菌中毒。BoNTs是生物恐怖主义的六种最高风险威胁因子之一，因为它们具有极高的效力和致命性，易于生产，需要长期的重症监护。马抗毒素的过敏反应发生率高，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。鉴于BoNTs的特殊毒性，抗毒素必须具有非常高的效力。以往的研究发现，没有一种单抗能够以所需的效力中和BONT，然而，结合与非重叠表位结合的单抗可以快速清除循环中的BONT，从而导致高效的BONT中和。我们已经产生了三种单抗的组合，它们结合了所有亚型的BoNT/A、B或E，并在体内导致了高度有效的BoNT中和。基于这种效力，卫生和公众服务部已将合同授予一家生物技术公司(XOMA(US)LLC)，以开发这些用于临床的单抗组合。BONT/A三种单抗组合已经进入第一阶段临床试验。BONT/B和BONT/E三种单抗组合正在生产cGMP，并将在2013-2014年前进入临床试验。这种方法的一个挑战是需要制造和质量控制的大量单抗(每个血清型3个)。这增加了产品的成本和复杂性，特别是如果mAbs都要组合在一起创建多血清型药物的话。我们建议的替代方案是为每种血清型创建一个三特异性单抗，其中包括三种单抗的结合位点。在这项工作中，我们假设可以产生这样的三特异单抗，它将具有与三种单抗组合相同的结合和BONT中和特性。在R21部分，我们将构建与三个非重叠的BONT/A表位结合的三特异性抗体(TsAbs)。将构建四个不同的TsAb，它们相对于抗体Fc的三个可变域中的每一个的位置不同。将从三个表位的亲和力、与BONT/A结合的化学计量比、稳定性以及表达和纯化的简易性等方面对每一株TSAb进行表征和优化。在小鼠中和试验中，将评估多个TSAb构建体的体内药代动力学(PK)以及BoNT/A清除和中和效力。活体研究将由我们的长期合作伙伴Luisa Cheng博士在美国农业部进行。这些研究将确定那些具有最佳性能的构建物，并将产生一种能够中和BoNT/A的单抗。在这项赠款的R33部分中，针对BoNT/A确定的最佳构建体将应用于其他三个BONT血清型，B、E和F。该项目的结果将是可以治疗99%以上的人类肉毒杆菌中毒病例的抗体。 公共卫生相关性：该项目的目标是产生高度有效和安全的抗体，用于预防和治疗由A型、B型、E型和F型肉毒神经毒素引起的肉毒杆菌中毒。BoNTs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，以治疗99%的人类肉毒杆菌中毒病例，这些病例将准备进行安全检测。

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.