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: 8608996
• 负责人: James D. Marks
• 金额: $ 114.01万
• 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 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8608996
• 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; 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; public health relevance; 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）：该项目的目的是开发一种高度有效的抗毒素，以治疗和预防由F型肉毒杆菌神经毒素（BONT/F）引起的肉毒杆菌毒素。抗毒素将包括三个或四种人类或人源化的单克隆抗体（mAb）的组合，这些抗体（mAb）共同结合并中和七个BONT/F子型。 BONT是生物恐怖主义的六种最高风险威胁者之一，因为它们的极端效能和致死性，易于生产以及需要长时间的重症监护。治疗的主体是通过过度免疫马制造的马抗毒素。马抗毒素的亚毒素的效力降低了，过敏反应的显着发生率，短血清半衰期导致重新启动，这是挑战性的，不能预防性地给出。我们已经生成了三个mAb的组合，它们结合了由BONT/A，B或E测试的所有子角膜型结合，并在体内导致高度有效的BONT中和。结合三个mAb结合非重叠表位的表位，由于循环迅速清除BONT，导致高度有效的BONT中和。根据该效力，HHS已授予加利福尼亚州伯克利市Xoma（US）LLC的合同，以开发这些MAB组合供临床使用。 BONT/A MAB组合已在1期临床试验中完成给药。 BONT/B和BONT/E MAB组合将在2014年第二季度进行临床试验。BONT/F特别具有挑战性，因为有七种已知的亚型型，它们在氨基酸水平上相互差异高达33％。亚色型序列变化导致马抗毒素的效力降低，并使发现粘合和中和所有子色谱的mAb具有挑战性。在NIAID U01资金下，我们开发了一组铅mab，结合了所有或许多Bont/f per-persotypes。我们还表明，当这些mAb中的三个mAb与高亲和力与BONT/F亚型型结合时，会发生有效的体内中和。对于此ProJec，我们假设可以设计这些铅mAB，以提高BONT/F子型的亲和力和交叉反应性，然后发展为三个或四个MAB组合，从而有效地中和所有BONT/F SUBSEROTYPES。我们将通过完成以下特定目标来实现这一目标。 AIM 1。评估和优化单个铅mAb特性，以识别三个或四个mAb的铅组合，这些单克值结合并中和七个报告的BONT/F子系列型。 AIM 2。产生无毒的BONT/F域，并针对每个单独的mAb进行特定的测定。目标3。发展表达mAb的稳定细胞系。 AIM 4。使用两步色谱法进行初始过程研究并实现90％的纯抗体。该项目完成后，我们将拥有一个由三个或四个人类或人性化的mAB组成的临床前候选抗毒素，这些抗毒素具有可接受的人体组织交叉反应性，结合并中和所有七个BONT/F子型亚曲线型，并且具有表达这些抗体的稳定细胞系。将来，该项目中生产的稳定的CHO细胞系可以作为GMP制造和临床开发的硕士和工作细胞库迅速发展。