Trispecific monoclonal antibody for botulinum neurotoxin intoxication therapy

用于肉毒杆菌神经毒素中毒治疗的三特异性单克隆抗体

• 批准号: 8839870
• 负责人: James D. Marks
• 金额: $ 43.83万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-20 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8839870
• 关键词: Affinity; Allergic Reaction; Amino Acid Sequence; Antibodies; Antibody Therapy; Antitoxins; Award; Binding; Binding Sites; Biological Assay; Biotechnology; Bioterrorism; Blood Circulation; Bontoxilysin; Botulinum Toxin Type A; Botulism; Clinical; Clinical Trials; Contracts; Cyclic GMP; Drug Kinetics; Epitopes; Equus caballus; Event; FDA approved; Funding; Goals; Grant; Half-Life; Human; Immunoglobulin Variable Region; Incidence; Individual; Intensive Care; Intoxication; Monoclonal Antibodies; Mus; Pharmaceutical Preparations; Phase I Clinical Trials; Positioning Attribute; Prevention; Production; Property; Proteins; Quality Control; Recombinant Antibody; Relative (related person); Serotyping; Serum; Testing; Therapeutic Effect; Toxic effect; Toxin; Work; abstracting; base; biothreat; botulinum toxin type B; botulinum toxin type E; cost; cost effective; high risk; human disease; in vivo; neutralizing monoclonal antibodies; safety testing; stoichiometry

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.

抽象的 该项目的目标是产生高效的三特异性抗体，用于预防和预防 治疗由肉毒神经毒素 (BoNT) 血清型 A、B、E 和 F 引起的肉毒杆菌中毒。BoNT 是六种血清型之一 由于其极高的效力和杀伤力、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 半衰期会导致再中毒，给药具有挑战性，并且不能预防性给药。鉴于 BoNT 具有非凡的毒性，抗毒素必须具有非常高的效力。之前的研究发现，没有 单一 mAb 以必要的效力中和 BoNT，但组合 mAb 结合不重叠 由于 BoNT 从循环中快速清除，表位导致高效的 BoNT 中和。 我们生成了三种 mAb 的组合，可结合 BoNT/A、B 或 E 的所有亚型，并产生 体内高效的 BoNT 中和作用。根据效力，HHS 已将合同授予 生物技术公司（XOMA（美国）LLC）开发这些单克隆抗体组合用于临床。荷兰银行/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大 需要生产和质量控制的 mAb 数量（每种血清型 3 个）。这增加了成本 和产品的复杂性，特别是如果所有单克隆抗体将被组合起来创建多血清型 药品。我们提出的替代方案是为每种血清型创建一个包含结合的三特异性单克隆抗体 三种单克隆抗体各自的位点。对于这项工作，我们假设可以生成这样的三特异性单克隆抗体 其与三种 mAb 组合具有相同的结合和 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 (TsAb) BoNT/A 表位。将构建四种不同的 TsAb，这些 TsAb 的不同之处在于三种抗体中的每一种的位置不同。 可变结构域相对于抗体Fc定位。每个 TsAb 将被表征和优化 关于对三个表位中每一个表位的亲和力、与 BoNT/A 结合的化学计量、稳定性和容易性 表达和纯化。将评估多种 TsAb 构建体的体内药代动力学 (PK) 和 小鼠中和测定中的 BoNT/A 清除率和中和效力。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 构建具有最佳特性的结构，并将产生有效中和 BoNT/A 的单一 mAb。在R33中 这笔拨款的一部分，为 BoNT/A 确定的最佳构建体将应用于其他三种 BoNT 血清型， B、E 和 F。该项目的成果将是能够治疗 99% 以上的人类肉毒中毒病例的抗体。