Development of Therapeutic Pan-Filovirus Macaque Monoclonal Antibodies

治疗性泛丝状病毒猕猴单克隆抗体的开发

• 批准号: 8496702
• 负责人: Sven G Enterlein
• 金额: $ 25.43万
• 依托单位: INTEGRATED BIOTHERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-27 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8496702
• 关键词: Advanced Development; Affinity; Africa; Antibodies; Antibody Affinity; Antibody Formation; Antibody Specificity; Antibody-Producing Cells; Antigens; B-Lymphocytes; Binding; Biological Assay; Biological Response Modifier Therapy; Biological Warfare; Blood; Blood specimen; Case Fatality Rates; Categories; Cell Culture Techniques; Cells; Centers for Disease Control and Prevention (U.S.); Clinical Trials; Code; Collection; Data; Democratic Republic of the Congo; Development; Disease Outbreaks; Distant; Dose; Dyes; Engineering; Enzyme-Linked Immunosorbent Assay; Family; Fc domain; Filovirus; Frankfurt-Marburg Syndrome Virus; Freezing; Genes; Globulins; Glycoproteins; Goals; Heterophile Antibodies; Human; Immune system; Immunization; Immunoglobulin G; Individual; Infection; Intervention; Lead; Legal patent; Length; Licensing; Life; Light; Lymphocyte; Macaca; Macaca mulatta; Mammalian Cell; Masks; Mediating; Messenger RNA; Microscopic; Minor; Modeling; Modification; Monkeys; Monoclonal Antibodies; Mucins; Mus; National Security; Neutralization Tests; Patients; Phage Display; Pharmaceutical Preparations; Phase; Preparation; Prevention; Primates; Production; Proteins; Protocols documentation; Public Health; Publishing; RNA; Reading; Reporting; Respiratory syncytial virus; Rodent; Secondary Immunization; Serum; Small Business Innovation Research Grant; Specificity; Sudan; Surface; System; Technology; Testing; Therapeutic; Therapeutic antibodies; Vaccinated; Vaccination; Vaccines; Viral Antigens; Viral Hemorrhagic Fevers; Virus; Virus-like particle; Work; antigen binding; base; disorder prevention; effective therapy; efficacy evaluation; expression vector; in vitro Assay; in vivo; innovation; member; miniaturize; mouse model; nonhuman primate; novel; novel strategies; pathogen; pre-clinical; prophylactic; protective efficacy; receptor binding; response; screening; symposium; therapeutic development

DESCRIPTION (provided by applicant): Filoviruses, Ebola (EBOV) and Marburg (MARV), are causative agents of sever hemorrhagic fever in humans with case-fatality rates exceeding 88%. Filoviruses, designated as category A select agents by the Centers for Disease Control and Prevention, are considered potential biowarfare agents and therefore a serious threat to public health and national security. Furthermore, outbreaks in Africa have been on the rise in the past 15 years. There are currently no vaccines or therapeutics available for filovirus hemorrhagic fever. Cellular entry of filoviruses is mediated by the envelope glycoprotein (GP) which is also known to be the primary protective antigen. The main species causing natural outbreaks are Zaire and Sudan EBOV as well as Lake Victoria MARV. A major challenge in development of effective treatment against filoviruses is the extensive sequence diversity within the family. We have recently defined the minimal receptor binding region RBR and demonstrated higher level of homology within this region (>40% between Zaire and Marburg and >80% between Zaire and Sudan). Preliminary data presented in this application demonstrate that an antigen based on the Marburg RBR can protect mice against lethal challenge with Zaire EBOV, the first report of cross protection between these two distant members of filoviruses. Crystallographic studies show that the RBR is masked by the bulky mucin-like domain (MLD). Integrated Biotherapeutics (IBT) has developed engineered antigens for the three major species denoted GPDMuc that expose the RBR by deletion of MLD and demonstrated induction of cross-species antibodies using this antigen. Furthermore, antibody response to this antigen was shown to correlate with protection from lethal challenge in nonhuman primate. In this proposal, we will use these novel antigens along with a powerful and innovative antibody discovery technology (CellSpot"), developed by Trellis Bioscience, to identify natural macaque antibodies induced by vaccination against the three filovirus species. CellSpot" is a robust and highly miniaturized platform for identification of secreted antibodies from single lymphocytes with the power to select for specificity and affinity against multiple antigens. The technology was successfully used to isolate a rare, cross-strain human antibody to Respiratory Syncytial Virus (RSV) with a 1 pM affinity that is licensed to Medimmune and is now being prepared for human clinical trials. In this project, we propose to use CellSpot to isolate therapeutic antibodies reactive to Zaire, Sudan, and Marburg viruses. The proposal is outlined in three Specific Aims. In Specific Aim 1 we will vaccinate rhesus macaques with filovirus-like particles (VLPs) followed by boosts with purified GPDMuc. Blood will be collected and the serum will be analyzed for a successful cross-species humoral response to GPDMuc (ELISA). In Specific Aim 2 PMBCs of reactive monkeys will be cultivated and analyzed for high-affinity antibodies in the CellSpot platform optimized for antibodies from nonhuman primates. RNA from B cell clones that produce cross-species binding antibodies will be extracted and the heavy and light chains will be cloned into expression vectors for small-scale production in mammalian cells. These candidate antibodies will be screened for antigen binding in a sensitive electrochemiluminescence-based (MSD) and other in vitro assays to identify candidates for testing with live viruses. In Specific Aim 3 we will evaluae the neutralizing activity of the ten best candidates (i.e. highest affinity) in cell culture using ive filoviruses. The 3-5 most promising antibodies will then tested in the lethal challenge mouse models of Ebola and Marburg infection. It is our goal to identify 1-3 antibodies with cross-species neutralizing activity. Upon successful completion of this Phase I SBIR we envision a Phase II focused on demonstration of protective efficacy in NHP models and standard pre-clinical development activities in preparation for regulatory filing.

描述（由申请人提供）：FILOVIRES，EBOLA（EBOV）和MARBURG（MARV）是人类严重出血热的致病药物，病例竞争率超过88％。疾病控制和预防中心被指定为特定代理的丝状病毒被认为是潜在的生物劳资代理商，因此对公共卫生和国家安全构成了严重威胁。此外，在过去的15年中，非洲的爆发一直在增加。目前尚无疫苗或治疗剂可用于丝状病毒出血热。丝状病毒的细胞进入是由包膜糖蛋白（GP）介导的，该糖蛋白（GP）也被称为主要保护性抗原。引起自然暴发的主要物种是Zaire和Sudan Ebov以及Victoria Marv湖。开发有效治疗的犯罪病毒的主要挑战是家庭内部的广泛序列多样性。我们最近定义了最​​小的受体结合区域RBR，并在该区域内表现出更高水平的同源性（在Zaire和Marburg之间> 40％，Zaire和Sudan之间的同源性较高）。本申请中提供的初步数据表明，基于Marburg RBR的抗原可以保护小鼠免受Zaire Ebov的致命挑战，这是FILOVIRASS这两个遥远成员之间的交叉保护的第一个报告。晶体学研究表明，RBR被笨重的粘蛋白样结构域（MLD）掩盖。综合生物治疗药（IBT）为三种主要物种开发了工程化的抗原，该物种表示GPDMUC，该物种通过删除MLD来暴露RBR，并证明了使用该抗原诱导了跨物种抗体。此外，对这种抗原的抗体反应显示与非人类灵长类动物的致命挑战有关。 In this proposal, we will use these novel antigens along with a powerful and innovative antibody discovery technology (CellSpot"), developed by Trellis Bioscience, to identify natural macaque antibodies induced by vaccination against the three filovirus species. CellSpot" is a robust and highly miniaturized platform for identification of secreted antibodies from single lymphocytes with the power to select for specificity and affinity against multiple抗原。该技术已成功地用于隔离一种罕见的，跨加工的人类抗体，其呼吸道合胞病毒病毒（RSV），其1 pm亲和力已获得许可，该亲和力已获得麦克膜元的许可，现在正在为人类的临床试验做好准备。在这个 项目，我们建议使用细胞点将治疗性抗体分离为Zaire，Sudan和Marburg病毒。该提案以三个具体目标概述。在特定的目标1中，我们将用丝状病毒样颗粒（VLP）接种恒河猕猴，然后用纯化的GPDMUC促进。将收集血液，并分析血清，以成功地跨物种对GPDMUC（ELISA）的体液反应。在特定目标中，将培养反应性猴子的2 pmbcs，并分析针对非人类灵长类动物的抗体优化的细胞点平台中的高亲和力抗体。将提取产生跨物种结合抗体的B细胞克隆中的RNA，并将重链和轻链克隆到表达载体中，以在哺乳动物细胞中进行小规模产生。这些候选抗体将在敏感的电化学（MSD）和其他体外测定中进行筛选，以识别用于使用活病毒测试的候选者的抗原结合。在特定的目标3中，我们将使用IVE FILOVIRES评估十种最佳候选者（即最高亲和力）的中和活性。然后，最有前途的抗体将在埃博拉和马堡感染的致命挑战小鼠模型中进行测试。我们的目标是识别具有中和活性的1-3种抗体。成功完成此阶段I SBIR后，我们设想了II期，重点是在NHP模型和标准的临床前开发活动中证明保护性疗效，以准备监管申请。