Receptor-Based Therapeutics for Enterotoxins

描述（由申请人提供）：细菌性肠毒素是一类被称为超级抗原（sag）的蛋白质的成员，具有致命活性，因为它们引发大量T细胞活化，导致炎症分子（如tnf - α）的释放。大多数已知的细菌sag是由金黄色葡萄球菌和化脓性链球菌表达的。这些包括生物恐怖主义的精选制剂，如葡萄球菌肠毒素B (SEB)，已被美国军方视为致残和致命的主要威胁。sag通过将T细胞受体v β区与另一个细胞上的II类MHC分子交联来激活T细胞。拟议的项目将开发可以中和肠道毒素的可溶性v β受体，并将涉及四名科学家的实验室，他们在sag的结构，功能和临床研究方面拥有丰富的经验：伊利诺伊大学的David Kranz；帕特里克·施里弗特，明尼苏达大学；罗伊·马里乌扎，美国马里兰州；西娜·巴伐利，美国陆军。该项目的受体工程部分将使用酵母展示技术生成针对一组sag的高亲和力拮抗剂（按优先顺序：SEB、TSST-1、SEC3、SpeA、SpeC）。根据预测SEB对人类的低LD50值（约1 μ g），很可能需要低皮摩尔亲和力的药物来有效中和。在之前的研究中，我们发现酵母修饰的高亲和力可溶性受体结构域（12kda）抑制了SEB和SEC3的体外活性。作为Vbeta:SAg相互作用可以被设计成低皮摩尔亲和力的证据，我们最近生成了一组SEB KD值为40至90 pM的Vbeta8突变体。目前的项目将进一步开发这些和其他可溶性Va受体作为治疗药物。具体目的是：1)设计和表征与sag高亲和力结合的vβ区：SEB， TSST-1, SEC3， SpeA和SpeC； 2)从目标1中生成vβ免疫球蛋白融合物，并表征其在小鼠中的结合特性和血清寿命；3)测试可溶性vβ - ig融合物在体外抑制sag活性和在动物模型中中和毒素的能力。这些模型将包括“人源化”的II类MHC/CD4转基因小鼠（包括气溶胶暴露模型）和兔子，它们在症状和毒性方面类似于人类sag介导的疾病。这里开发的策略是设计非常高亲和力的中和受体，并增加它们的血清寿命，应该直接转化为开发对抗其他毒素的拮抗剂，这些毒素被认为是生物恐怖主义的潜在代理人。

DESCRIPTION (provided by applicant): Bacterial enterotoxins are members of a class of proteins known as superantigens (SAgs) that have lethal activity because they elicit massive T cell activation, leading to release of inflammatory molecules such as TNF-alpha. Most of the known bacterial SAgs are expressed by Staphylococcus aureus and Streptococcus pyogenes. These include select agents of bioterrorism such as staphylococcal enterotoxin B (SEB) that has been considered by the U.S. military as a major threat for incapacitation and lethality. SAgs activate T cells by cross-linking T cell receptor Vbeta regions with class II MHC molecules on another cell. The proposed project will develop soluble Vbeta receptors that can neutralize enterotoxins and will involve the laboratories of four scientists that have extensive experience in structural, functional, and clinical studies of SAgs: David Kranz, University of Illinois; Patrick Schlievert, U. Minnesota; Roy Mariuzza, U. Maryland; Sina Bavari, U.S. Army. The receptor engineering components of the project will use yeast display technology to generate high-affinity antagonists against a panel of SAgs (in the order of priority: SEB, TSST-1, SEC3, SpeA, SpeC). Based on the low LD50 values (approximately 1 mu g) of SEB predicted for humans, it is very likely that low picomolar affinity agents will be required for effective neutralization. In previous work, we snowed that high-affinity, soluble receptor domains (12 KDa) engineered by yeast display inhibited the in vitro activity of SEB and SEC3. As evidence that Vbeta:SAg interactions can be engineered to low picomolar affinities, we recently generated a panel of Vbeta8 mutants with KD values for SEB from 40 to 90 pM. The present project will further develop these, and other soluble Va receptors, as therapeutics. The specific aims are to: 1) To engineer and characterize Vbeta regions that bind with high-affinity to SAgs: SEB, TSST-1, SEC3, SpeA, and SpeC, 2) To generate Vbeta immunoglobulin fusions of the agents from aim 1 and to characterize their binding properties and serum lifetimes in mice, 3) To test the ability of soluble Vbeta-Ig fusions to inhibit activity of SAgs in vitro and to neutralize the toxins in animal models. The models will include "humanized" class II MHC/CD4 transgenic mice (including an aerosol model of exposure) and rabbits, which resemble human SAg-mediated diseases in symptoms and toxicity. The strategies developed here to engineer very high-affinity neutralizing receptors and to increase their serum lifetimes should be directly translatable to the development of antagonists against other toxins that are considered potential agents of bioterrorism.