Generation of therapeutic antibodies to serotype F botulism

针对血清型 F 肉毒杆菌中毒的治疗性抗体的产生

• 批准号: 8790945
• 负责人: James D. Marks
• 金额: $ 114.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8790945
• 关键词: Affinity; Allergic Reaction; Amino Acid Sequence; Amino Acids; Antibodies; Antitoxins; Award; Binding; Biological Assay; Biotechnology; Bioterrorism; Blood Circulation; Bontoxilysin; Botulinum Toxin Type A; Botulism; Cell Line; Characteristics; Chinese Hamster Ovary Cell; Chromatography; Clinical; Clinical Trials; Contracts; Cyclic GMP; Development; Dose; Effectiveness; Engineering; Epitopes; Equus caballus; Funding; Future; Generations; Goals; Half-Life; Health; Human; Incidence; Individual; Intensive Care; Investigation; Lead; Monoclonal Antibodies; National Institute of Allergy and Infectious Disease; Phase I Clinical Trials; Prevention; Process; Production; Reporting; Safety; Serotyping; Serum; Testing; Therapeutic antibodies; Toxic effect; Variant; Work; assay development; base; biothreat; botulinum toxin type B; botulinum toxin type E; cell bank; cross reactivity; high risk; human disease; human tissue; humanized monoclonal antibodies; improved; in vivo; neutralizing monoclonal antibodies; pre-clinical; stable cell line

DESCRIPTION (provided by applicant): The goal of this project is to develop a highly potent antitoxin for the treatment and prevention of botulism caused by type F botulinum neurotoxin (BoNT/F). The antitoxin will consist of a combination of three or four human or humanized monoclonal antibodies (mAbs) which together bind to and neutralize the seven BoNT/F sub-serotypes. 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. The mainstay of treatment is equine antitoxin, made by hyperimmunizing horses. Equine antitoxin has a reduced potency for sub-serotypes, a significant incidence of allergic reactions, a short serum half-life leading to reintoxication, is challenging to administer, and cannot be given prophylactically. We have generated combinations of three mAbs that bind all sub-serotypes tested of BoNT/A, B, or E and result in highly potent BoNT neutralization in vivo. Combining three mAbs binding non-overlapping epitopes leads to highly potent BoNT neutralization due to rapid clearance of BoNT from the circulation. Based on the potency, HHS has awarded contracts to XOMA (US) LLC, Berkeley, CA to develop these mAb combinations for clinical use. The BoNT/A mAb combination has completed dosing in a Phase 1 clinical trial. The BoNT/B and BoNT/E mAb combinations will enter clinical trials by second quarter 2014. BoNT/F is particularly challenging, as there are seven known sub-serotypes, which differ from each other by up to 33% at the amino acid level. Sub-serotype sequence variation results in reduced potency of equine antitoxin and makes finding mAbs that bind and neutralize all sub-serotypes challenging. Under NIAID U01 funding, we have developed a panel of lead mAbs binding all or many of the BoNT/F sub- serotypes. We have also shown that when three of these mAbs bind with high affinity to a BoNT/F sub- serotype, potent in vivo neutralization occurs. For this projec, we hypothesize that these lead mAbs can be engineered to improve affinity and cross reactivity for the BoNT/F sub-serotypes and then developed into a three or four mAb combination that potently neutralizes all BoNT/F sub-serotypes. We will accomplish this by completing the following specific aims. Aim 1. Evaluate and optimize individual lead mAb characteristics to identify lead combinations of three or four mAbs that bind and neutralize the seven reported BoNT/F sub-serotypes. Aim 2. Generate non-toxic BoNT/F domains and assays specific for each individual mAb. Aim 3. Develop stable cell lines that express mAbs. Aim 4. Conduct initial process investigations using two-step chromatography and achieve 90% pure antibody. At the completion of this project, we will have a preclinical lead candidate BoNT/F antitoxin consisting of three or four human or humanized mAbs that have acceptable human tissue cross reactivity, bind and neutralize all seven BoNT/F sub-serotypes, and have stable cell lines that express these antibodies. The stable CHO cell lines produced in this project can be rapidly developed in the future as master and working cell banks for GMP manufacture and clinical development.

描述（由申请人提供）：该项目的目标是开发一种高效抗毒素，用于治疗和预防 F 型肉毒神经毒素（BoNT/F）引起的肉毒杆菌中毒。该抗毒素将由三四种人或人源化单克隆抗体 (mAb) 组合而成，这些抗体共同结合并中和七种 BoNT/F 亚血清型。 BoNT 因其极高的效力和杀伤力、易于生产以及需要长期重症监护而成为生物恐怖主义六种最高风险的威胁因子之一。治疗的主要方法是马抗毒素，由高度免疫的马产生。马抗毒素对亚血清型的效力降低，过敏反应发生率显着，血清半衰期短，导致再中毒，给药具有挑战性，并且不能预防性给药。我们已经生成了三种 mAb 的组合，它们可以结合 BoNT/A、B 或 E 测试的所有亚血清型，并在体内产生高效的 BoNT 中和作用。由于 BoNT 从循环中快速清除，结合三种结合非重叠表位的 mAb 可实现高效的 BoNT 中和。根据效力，HHS 已向加利福尼亚州伯克利市的 XOMA (US) LLC 授予合同，开发这些单克隆抗体组合用于临床。 BoNT/A mAb 组合已在 1 期临床试验中完成给药。 BoNT/B 和 BoNT/E mAb 组合将于 2014 年第二季度进入临床试验。BoNT/F 特别具有挑战性，因为有七种已知的亚血清型，它们在氨基酸水平上的差异高达 33% 。亚血清型序列变异导致马抗毒素的效力降低，并且使得寻找结合和中和所有亚血清型的单克隆抗体具有挑战性。在 NIAID U01 的资助下，我们开发了一组可结合所有或多种 BoNT/F 亚血清型的先导单克隆抗体。我们还表明，当其中三种 mAb 以高亲和力与 BoNT/F 亚血清型结合时，会发生有效的体内中和作用。对于这个项目，我们假设这些先导单克隆抗体可以经过改造以提高 BoNT/F 亚血清型的亲和力和交叉反应性，然后开发成三或四种单克隆抗体组合，可有效中和所有 BoNT/F 亚血清型。我们将通过完成以下具体目标来实现这一目标。目标 1. 评估和优化单个先导 mAb 特性，以确定结合并中和报告的七种 BoNT/F 亚血清型的三种或四种 mAb 的先导组合。目标 2. 生成针对每种单克隆抗体的无毒 BoNT/F 结构域和检测方法。目标 3. 开发表达 mAb 的稳定细胞系。目标 4. 使用两步色谱法进行初步工艺研究并获得 90% 纯度的抗体。该项目完成后，我们将拥有一种临床前主要候选 BoNT/F 抗毒素，由三到四种人或人源化 mAb 组成，这些单克隆抗体具有可接受的人体组织交叉反应性，结合并中和所有七种 BoNT/F 亚血清型，并具有稳定的稳定性。表达这些抗体的细胞系。本项目生产的稳定CHO细胞系未来可快速发展为GMP生产和临床开发的主细胞库和工作细胞库。