Optimization of mAbs to staphylococcal enterotoxin B for treatment

该应用程序将汇集不同研究人员的专业知识和最新技术水平 平台技术，旨在优化葡萄球菌肠毒素B的单克隆抗体（mAb） (SEE)供人类使用。除了产生一种新的治疗试剂的直接好处外，这项工作将 研究与毒素中和和清除有关的几个假设。基础工作 我们提出假设是，高亲和力抗SEB mAb最适合于治疗用途，因为 少量的mAb将足以成功地与已经结合的SEB竞争。我们将调查，如果 人单克隆抗体在中和毒素能力上是不同的，并且是上级的。 具有对FcR结合作用的C-末端的工程改造将改变体内毒素清除。过去 在资助期内，针对SEB的11种mAb已被亚克隆、表征并在鼠动物模型中进行了测试， SEB诱导休克。开发了一种高灵敏度的捕获ELISA，这将使我们能够在建议的 测定中毒小鼠血清中SEB毒素的实验。此外，SEB生产的普遍性 S.金黄色葡萄球菌菌株中的重要序列变异性。金黄色 将被考虑的因素。已经选择了三种IgG 1候选mAb用于靶向 AB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。在动物模型和人类T细胞中记录了保护作用。 刺激测定。此外，克隆了mAb的重链和轻链可变区。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， 提出产生新的人SEB特异性mAb，一种涉及遗传修饰的小鼠， 表达人类可变区，另一个我们将使用艾哈迈德/威尔逊博士的技术， 直接从原发性S.金黄色葡萄球菌感染。最后，在目标3中， 与Ravetch博士合作设计mAb的FC结合部分，并测试有趣的 假设mAb组合增强中和和清除。

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.