RECOMBINANT SHIGA-TOXIN-SPECIFIC HUMAN ANTIBODIES

重组志贺毒素特异性人类抗体

• 批准号: 6291566
• 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 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6291566
• 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; 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.

该建议是对RFA的回应：DK-00-005“食源性疾病，胃肠道和肾脏并发症”，特别解决了“干预措施和/或临床策略的开发”，以防止大肠杆菌0157：H7相关疾病的并发症。出血性结肠炎与HUS的发展之间的关键间隔”。该提案的目的是开发一种有效的基于免疫的配方，可以安全地给有患有志贺 - 毒素（STX）相关溶血性尿毒症综合征（HUS）的儿童使用。该治疗的目标种群包括儿童，该儿童可以直接或间接地归因于STX。目前尚无对HUS的有效治疗或预防。在该提案中，我们证明了我们在单独的NIH奖励下生成的外源STX特异性人单克隆抗体（HU-MABS），在口服挑战6-12小时后，请保护Gnotobiotic Piglet免受STX介导的致命神经系统症状。大肠杆菌0157：H7。相反，在口腔挑战后的2-3天内，用安慰剂治疗的小猪会出现血管介导的致命神经系统症状。在此应用程序中，我们希望在真核系统中表达这些抗STX HU-mab中的几个。这将使我们能够大量生产它们（特定目的1）和不同形式的形式，例如同型变体（特定AIM 2）和Fab碎片（AIM 3），以确定哪种形式可以在体外提供最有效的保护和体内（特定目标4）。我们预计最终产品将含有Activation STX1和STX2的A和B亚基的HU-MAB鸡尾酒（特定AIM 4）。鉴于我们的初步数据，我们相信专门设计且高度集中的HU-MAB将在保护有HUS风险的儿童中安全有效。我们认为重组HU-mab（作为整个分子或Fabs）将同样有效。重组Hu-mabs将大大提高这些试剂的生产效率，并使它们用于临床使用。与其他来源（例如多克隆抗体或鼠/嵌合mabs）相比，Hu-mab具有更长的半衰期，对靶标的更好的亲和力，更高的效力，需要较低的剂量，更安全，显然是未来的波浪。我们有信心，在五年支持期结束时，我们将在真核系统中表达，这是一个充分表征的有效重组STX特异性HU-MAB的小组，准备根据FDA指南制定临床评估。