RECOMBINANT SHIGA-TOXIN-SPECIFIC HUMAN ANTIBODIES

重组志贺毒素特异性人类抗体

• 批准号: 6609700
• 负责人: SAUL r TZIPORI
• 金额: $ 33.4万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6609700
• 关键词: CHO cells; Escherichia coli 0157:H7; Escherichia coli infections; HeLa cells; SDS polyacrylamide gel electrophoresis; antibacterial antibody; drug design /synthesis /production; drug screening /evaluation; electroporation; enzyme linked immunosorbent assay; hemolytic anemia; hybridomas; immunoglobulin G; immunotherapy; laboratory mouse; molecular cloning; monoclonal antibody; neutralizing antibody; polymerase chain reaction; recombinant proteins; renal failure; shiga toxin; swine; synthetic antigens; transfection /expression vector; western blottings

This proposal is in response to RFA: DK-00-005 "Foodborne Illness, Gastrointestinal and Renal Complications", specifically addressing "Development of interventions and/or clinical strategies to prevent complications of E. coli 0157:H7-related illnesses during the initial critical interval between the occurrence of hemorrhagic colitis and the development of HUS". The goal of this proposal is to develop an effective immune-based formulation which can be administered safely to children at risk of developing Shiga-toxin (Stx)-related hemolytic uremic syndrome (HUS). The target populations for this treatment include children in whom the disease can directly or indirectly be attributed to Stx. Currently there is no effective treatment or prevention for HUS. In this proposal we demonstrate that exogenous Stx-specific human monoclonal antibodies (Hu-mAbs) which we have generated under a separate NIH award, protect gnotobiotic piglets against Stx-mediated fatal neurological symptoms when administered 6-12 hours after oral challenge with E. coli 0157:H7. In contrast, piglets treated with placebo develop vascular-mediated fatal neurological symptoms within 2-3 days after the oral challenge. In this application we wish to express several of these anti-Stx Hu-mAbs in an eukaryotic system. This will allow us to produce them in large quantities (Specific Aim 1) and in different forms, such as isotype-variants (Specific Aim 2) and Fab fragments (aim 3) to determine which form would provide the most effective protection in vitro and in vivo (Specific Aim 4). We anticipate the final product will contain a cocktail of Hu-mAbs active against the A and B subunits of Stx1 and Stx2 (Specific Aim 4). Given our Preliminary Data, we are confident that specifically designed and highly concentrated Hu-mAbs will be safe and effective in protecting children at risk of HUS. We believe recombinant Hu-mAbs, either as whole molecules or Fabs, will be equally effective. Recombinant Hu- mAbs will considerably improve the efficiency of production of these reagents and make them available for clinical use. In contrast to other sources, such as polyclonal antibodies or murine/chimeric mAbs, Hu- mAbs have longer half-life, better affinity for targets, higher potency, require a lower dose, are safer, and are clearly the wave of the future. We are confident that at the end of the five-year support period we will have expressed in an eukaryotic system, a fully characterized panel of effective recombinant Stx-specific Hu-mAbs, ready to be produced under FDA guidelines for clinical evaluation.

该提案是对 RFA：DK-00-005“食源性疾病、胃肠道和肾脏并发症”的回应，具体涉及“制定干预措施和/或临床策略，以预防大肠杆菌 0157:H7 相关疾病在初始阶段的并发症”出血性结肠炎发生与 HUS 发展之间的关键间隔”。该提案的目标是开发一种有效的基于免疫的制剂，可以安全地给有患志贺毒素（Stx）相关溶血性尿毒症综合征（HUS）风险的儿童使用。这种治疗的目标人群包括疾病可直接或间接归因于 Stx 的儿童。目前HUS尚无有效的治疗或预防方法。在本提案中，我们证明，我们根据 NIH 的单独奖项生产的外源 Stx 特异性人单克隆抗体 (Hu-mAb)，在口服大肠杆菌攻击 6-12 小时后，可以保护无菌仔猪免受 Stx 介导的致命神经系统症状。大肠杆菌 0157:H7。相比之下，接受安慰剂治疗的仔猪在口服攻击后 2-3 天内出现血管介导的致命神经症状。在此应用中，我们希望在真核系统中表达其中几种抗 Stx Hu-mAb。这将使我们能够以不同的形式大量生产它们（具体目标 1），例如同种型变体（具体目标 2）和 Fab 片段（目标 3），以确定哪种形式可以提供最有效的体外保护和体内（具体目标 4）。我们预计最终产品将包含针对 Stx1 和 Stx2 的 A 和 B 亚基具有活性的 Hu-mAb 混合物（具体目标 4）。根据我们的初步数据，我们相信专门设计的高浓度 Hu-mAb 将安全有效地保护面临 HUS 风险的儿童。我们相信重组 Hu-mAb，无论是整个分子还是 Fab，都将同样有效。重组人单克隆抗体将大大提高这些试剂的生产效率并使其可用于临床。与多克隆抗体或鼠/嵌合单克隆抗体等其他来源相比，Hu-单克隆抗体具有更长的半衰期、更好的靶点亲和力、更高的效力、更低的剂量、更安全，并且显然是未来的趋势。我们相信，在五年支持期结束时，我们将在真核系统中表达，这是一组完全表征的有效重组 Stx 特异性 Hu-mAb，准备好根据 FDA 指南进行生产以进行临床评估。