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 该项目的目的是生成用于预防和预防的高度特异性抗体 肉毒杆菌神经毒素（BONT）血清型A，B，E和F的治疗。 最高风险的生物恐怖威胁因素，由于其极端的效力和致死性，易于生产性和 需要长时间的重症监护。马抗毒素的过敏反应发生率很高，短血清 半衰期会导致重新施加，具有挑战性的管理，不能预防性给予。鉴于 抗毒素的抗毒毒素的特殊毒性必须具有很高的效力。以前的研究发现没有 单个mAb用必要的效力中和骨，但是结合了mab的结合非重叠 由于循环的快速清除BONT，表位导致高度有效的BONT中和。 我们已经生成了三个mab的组合，它们结合了bont/a，b或e的所有亚型，并导致 体内高度有效的骨中和。根据效力，HHS已将合同授予 生物技术公司（XOMA（US）LLC）开发这些MAB组合以供临床使用。 Bont/a 三个mAb组合已进入1期临床试验。 BONT/B和BONT/E三mAb组合 正在CGMP制造中，并将在2013 - 2014年之前进行临床试验。这种方法的挑战是很大 需要制造和控制质量的mAB数量（每种血清型3）。这增加了费用 和产品的复杂性，尤其是如果将单元单击全部组合起来以创建多种形式 药品。我们提出的选择是为包含结合的每个血清型创建一个单个特异性mAb 三个mab中每个位置的位置。对于这项工作，我们假设可以生成这种特异性单元 它具有与三个mAb组合相同的结合和骨中和中和的特性。 在R21部分中，我们将构建三个非重叠的二级特异性抗体（TSAB） BONT/A表位。将构建四个不同的tsabs 可变域相对于抗体FC的位置。每个TSAB都将被表征和优化 关于三个表位中的每个表位的亲和力，结合到BONT/A的化学计量，稳定性 表达和纯化。将评估多个TSAB构建体的体内药代动力学（PK）和 小鼠中和测定中的BONT/A间隙和中和效力。体内研究将是 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 具有最佳特性的构造，将产生一个单个mab，该mab有效地中和bont/a。在R33中 该赠款的一部分，标识的BONT/A的最佳结构将应用于其他三种BONT血清型， B，E和F。该项目的结果将是将超过99％的人类肉毒杆菌病例治疗的抗体。