Receptor-Based Therapeutics for Enterotoxins

描述(申请人提供)：细菌肠毒素是一类被称为超抗原(SAG)的蛋白质的成员，具有致死活性，因为它们能诱导大量T细胞激活，导致释放炎症分子，如肿瘤坏死因子-α。已知的大部分细菌下垂由金黄色葡萄球菌和化脓性链球菌表达。其中包括精选的生物恐怖主义制剂，如葡萄球菌肠毒素B(SEB)，它被美国军方视为对丧失能力和致命性的主要威胁。SAGS通过将T细胞受体Vbeta区与另一个细胞上的II类MHC分子交联来激活T细胞。拟议中的项目将开发能够中和肠道毒素的可溶性Vbeta受体，并将涉及四位在SAG的结构、功能和临床研究方面拥有丰富经验的科学家的实验室：伊利诺伊大学的David Kranz；明尼苏达州大学的Patrick Schlievert；美国马里兰州的Roy Mariuzza；美国陆军的Sina Bavari。该项目的受体工程组件将使用酵母展示技术来产生针对一组SAG的高亲和力拮抗剂(按优先顺序为：SEB、TSST-1、SEC3、SPEA、SPEC)。根据SEB对人体预测的低LD50值(约1微克)，很可能需要低皮摩尔亲和力的试剂才能有效中和。在以前的工作中，我们发现酵母展示的高亲和力的可溶性受体结构域(12 KDa)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，我们最近产生了一组Vbeta8突变体，其SEB的Kd值从40到90 PM本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。其具体目的是：1)设计和鉴定与SAGS高亲和力结合的Vbeta区域：SEB、TSST-1、SEC3、SPEA和SPEC；2)从Aim 1产生这些制剂的Vbeta免疫球蛋白融合，并鉴定它们的结合特性和小鼠血清寿命；3)测试可溶性Vbeta-Ig融合在体外抑制SAGS活性的能力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(包括暴露在空气中的气雾剂模型)和兔子，它们在症状和毒性上类似于人类SAG介导性疾病。这里开发的设计非常高亲和力的中和受体并延长它们的血清寿命的策略应该可以直接转化为针对其他被认为是潜在的生物恐怖主义因子的毒素的拮抗剂的开发。

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.