Optimization of mAbs to staphylococcal enterotoxin B for treatment

这一应用将汇集不同调查人员的专业知识，并不合作最先进的技术 平台技术，目标是优化抗葡萄球菌肠毒素B的单抗 (见)供人类使用。除了产生一种新的治疗试剂的直接好处外，这项工作还将 研究与毒素中和和清除有关的几个假设。潜在的工作 我们建议的假设是高亲和力的抗SEB单抗最适合用于治疗，因为 少量的mAb将足以成功地与已结合的SEB竞争。我们将调查是否 人单抗在中和毒素的能力上有所不同并具有优势，此外，我们建议靶向 影响FCR结合的C末端的工程将改变体内的毒素清除。在过去 资助期11株抗SEB单抗已被亚克隆、鉴定并在小鼠动物模型中进行试验 SEB诱导休克。开发了一种高度灵敏的捕获酶联免疫吸附试验，将使我们能够提出 中毒小鼠血清中SEB毒素的检测实验。此外，SEB的产生也很普遍 已经建立了金黄色葡萄球菌菌株，并在不同的金黄色葡萄球菌中鉴定了重要的序列变异 这些菌株将被考虑在内。已选定三株lgG1候选单抗作为靶向 AB工程学。他们识别蛋白质C-末端部分的表位，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 刺激分析。此外，还克隆了单抗的重链可变区和轻链可变区。在目标一号中 我们建议与沙尔夫博士合作，通过亲和力成熟来优化这些单抗。在《目标2》中，我们 建议产生新的人类SEB特异性单抗，其中一个涉及一种转基因小鼠， 表达人类可变区，另一个我们将使用Ahmed/Wilson博士的技术来 直接克隆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mAbs的Fc结合部分，并测试有趣的 单抗结合可增强中和和清除作用的假设。

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.