Trispecific monoclonal antibody for botulinum neurotoxin intoxication therapy

摘要 该项目的目标是产生高效的三特异性抗体，用于预防和治疗 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 生物恐怖主义的最高风险威胁因子，因为它们具有极强的效力和杀伤力，易于生产，以及 需要长期的重症监护。马抗毒素过敏反应发生率高，血清短 半衰期导致再中毒，给药具有挑战性，不能预防性给予。给定 BoNTs的特殊毒性，抗毒素必须具有非常高的效力。以前的研究发现，没有 单一单抗以必要的效力中和BONT，但结合的单抗结合不重叠 由于快速清除循环中的BONT，表位可导致高度有效的BONT中和。 我们已经生成了三个mAb的组合，它们绑定了BONT/A、B或E的所有子类型，并导致 体内高效的BONT中和作用。根据效力，HHS已将合同授予一家 生物技术公司(XOMA(美国)有限责任公司)开发这些临床使用的单抗组合。BNOT/A 三种单抗组合已进入一期临床试验。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 需要生产和质量控制的单抗数量(每个血清型3个)。这增加了成本 以及产品的复杂性，特别是如果所有mAbs都要结合起来创建多血清型的话 毒品。我们建议的替代方案是为每个血清型创建一个包含结合的三特异单抗 三种单抗中每一种的结合部位。在这项工作中，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，我们将构建结合三个非重叠的三特异性抗体(TsAbs BONT/A表位。将构建四个不同的TsAb，它们相对于三个TsAb中的每一个的位置不同 可变区相对于抗体Fc定位。将对每个TSAB进行特色化和优化 关于三个表位中每一个的亲和力、与BONT/A结合的化学计量比、稳定性和易用性 表达和纯化。将评估多种TSAb构建体的体内药代动力学(PK)和 小鼠中和试验中的BoNT/A清除和中和效力。体内研究将是 由我们的长期合作伙伴路易莎·郑博士在美国农业部进行。这些研究将确定 构建具有最佳性能的结构，并将产生一种有效中和BONT/A的单抗。在R33中 作为这笔赠款的一部分，为BONT/A确定的最佳结构将应用于其他三种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。

Abstract 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 potently 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.