Optimization of mAbs to staphylococcal enterotoxin B for treatment

针对葡萄球菌肠毒素 B 的单克隆抗体的优化治疗

• 批准号: 8230239
• 负责人: Bettina Fries
• 金额: $ 57.43万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8230239
• 关键词: Affinity; Animal Model; B-Lymphocytes; Binding; Biological Assay; C-terminal; Collaborations; Dose; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Funding; Goals; Human; Immunoglobulin Constant Region; Immunoglobulin Variable Region; Infection; Knowledge; Light; Measures; Monoclonal Antibodies; Mus; Patients; Proteins; Reagent; Research Personnel; Serum; Shock; Staphylococcal Enterotoxin B; Staphylococcus aureus; T-Lymphocyte; Techniques; Technology; Testing; Therapeutic Uses; Time; Toxin; Treatment Protocols; Work; base; biodefense; cost; design; improved; in vivo; novel therapeutics; research study

This application will bring together the expertise of different investigators and incooperate state of the art platform technologies with the goal to optimize monoclonal antibodies (mAb) to Staphylococcal enterotoxin B (SEE) for human use. Beyond the immediate benefit of yielding a novel therapeutic reagent, this work will investigate several hypotheses that pertain to toxin-neutralization and clearance. The underlying working hypothesis of our proposal is that high affinity anti-SEB mAbs are best suited for therapeutic use because low amounts of mAb will be sufficient to successfully compete with already bound SEB. We will investigate if human mAbs differ and are superior in their ability to neutralize toxin, In addition we propose that targeted engineering of the C-terminal with effects on FcR binding will change toxin clearance in vivo. In the past funding period 11 mAbs to SEB have been subcloned, characterized and tested in murine animal model for SEB induced shock. A highly sensitive capture ELISA was developed that will allow us in proposed experiments to measure SEB toxin in serum of intoxicated mice. In addition the prevalance of SEB producing S. aureus strains has been established and identified important sequence variabilities in different S. aureus strains which will be taken into consideration. Three lgG1 candidate mAbs have been selected for targeted Ab engineering. They recognize epitopes in the C-terminal part of the protein, which is also the predominant epitope recognized by human B-cells. Protection was documented in animal models and human T-cell stimulation assays. In addition heavy and light chain variable regions of the mAbs were cloned. In Aim one we propose to optimize these mAbs by affinity maturation in collaboration with Dr. Scharff. In Aim 2 we propose to generate new human SEB apecific mAbs one involving a genetically modified mouse that expresses the human variable region and the other one we will use Drs. Ahmed/Wilson's technique to directly clone the variable region from patients with primary S. aureus infection. Finally in aim 3 we will engineer the FC binding portion of the mAbs in collaboration with Dr. Ravetch and test the intriguing hypothesis that combination of mAbs enhances neutralization and clearance.

该应用程序将汇集不同研究人员的专业知识并合作最先进的技术 旨在优化葡萄球菌肠毒素 B 的单克隆抗体 (mAb) 的平台技术 （参见）供人类使用。除了产生新型治疗试剂的直接好处之外，这项工作还将 研究与毒素中和和清除有关的几种假设。底层工作 我们提议的假设是高亲和力抗 SEB mAb 最适合治疗用途，因为 少量的 mAb 就足以成功地与已经结合的 SEB 竞争。我们将调查是否 人类单克隆抗体在中和毒素的能力方面有所不同且更优越，此外，我们建议靶向 影响 FcR 结合的 C 末端工程将改变体内毒素清除率。在过去 资助期间 11 个针对 SEB 的单克隆抗体已在小鼠动物模型中进行了亚克隆、表征和测试 SEB引起的冲击。开发了一种高度灵敏的捕获 ELISA，这将使我们能够 测量中毒小鼠血清中 SEB 毒素的实验。此外，SEB 生产的盛行 金黄色葡萄球菌菌株已建立并鉴定出不同金黄色葡萄球菌中重要的序列变异 将被考虑的菌株。已选择三种 IgG1 候选单克隆抗体用于靶向治疗 抗体工程。它们识别蛋白质 C 端部分的表位，这也是主要的 人类 B 细胞识别的表位。动物模型和人类 T 细胞中记录了保护作用 刺激试验。此外还克隆了单克隆抗体的重链和轻链可变区。在目标一 我们建议与 Scharff 博士合作，通过亲和力成熟来优化这些 mAb。在目标 2 中，我们 提议产生新的人类 SEB 特异性单克隆抗体，其中一种涉及转基因小鼠， 表示人类可变区，另一个我们将使用 Drs。艾哈迈德/威尔逊的技术 直接克隆原发性金黄色葡萄球菌感染患者的可变区。最后，在目标 3 中，我们将 与 Ravetch 博士合作设计 mAb 的 FC 结合部分并测试有趣的结果 假设单克隆抗体的组合可以增强中和和清除。