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 治疗由肉毒神经毒素（BoNT）血清型A、B、E和F引起的肉毒中毒。BONT是六个 生物恐怖主义的最高风险威胁剂，由于其极端的效力和致命性，易于生产， 需要长期的重症监护。马抗毒素过敏反应发生率高，血清短， 导致再中毒的半衰期，给药具有挑战性，并且不能无菌给药。鉴于 BoNT的毒性非常大，抗毒素必须具有非常高的效力。以前的研究发现， 单个mAb以必要的效力中和BoNT，然而组合mAb结合非重叠 由于BoNT从循环中的快速清除，表位导致高度有效的BoNT中和。 我们已经产生了三种mAb的组合，其结合BoNT/A、B或E的所有亚型，并导致 体内高效BoNT中和。根据效力，HHS已将合同授予一家 生物技术公司（XOMA（US）LLC）开发这些mAb组合用于临床用途。BoNT/A 三种mAb组合已进入1期临床试验。BoNT/B和BoNT/E三种mAb组合 正在进行cGMP生产，并将于2013 - 2014年进入临床试验。这种方法的一个挑战是 需要生产和质量控制的mAb数量（每种血清型3个）。这就增加了成本 和产品的复杂性，特别是如果单克隆抗体都要结合起来，创造一个多血清型 药我们提出的替代方案是为每种血清型创建一个单一的三特异性mAb， 三种mAb中每一种的位点。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中和性质。 在R21部分中，我们将构建结合三个非重叠区的三特异性抗体（TsAb）。 BoNT/A表位。将构建四种不同的TsAb，它们在三种TsAb中的每一种的位置上不同。 可变结构域相对于抗体Fc定位。将对每种TsAb进行表征和优化 关于对三个表位中的每一个的亲和力、与BoNT/A结合的化学计量、稳定性和容易性， 表达和纯化。将评价多种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 由我们的长期合作者Luisa Cheng博士在美国农业部进行。这些研究将确定那些 构建体具有最佳性质，并将产生有效中和BoNT/A的单一mAb。在R33 作为该拨款的一部分，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。

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.