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Receptor-Based Therapeutics for Enterotoxins

基于受体的肠毒素治疗

基本信息

批准号：
7541430
负责人：
David M. Kranz
金额：
$ 29.67万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-02-01 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7541430
关键词：
Aerosols Affinity Animal Model Animals Binding Binding Sites Biochemical Biochemistry Bioterrorism Blood specimen Body Temperature Cell Line Cell Separation Cells Clinical Research Communicable Diseases Development Disease Drug Kinetics Engineering Enterotoxins Family Flow Cytometry Generations Genetic Engineering Goals Histocompatibility Antigens Class II Human Illinois Immunoglobulin G Immunoglobulins In Vitro Inflammatory Knowledge Laboratories Lethal Dose 50 Libraries MHC Class II Genes Maryland Measures Mediating Medical Research Military Personnel Minnesota Modeling Molecular Monitor Mus Mutagenesis Oryctolagus cuniculus Property Proteins Research Institute Scientist Serum Speed Staphylococcal Enterotoxin B Staphylococcus aureus Streptococcus pyogenes Streptococcus pyogenes SpeA protein Structure Superantigens Surface Symptoms T-Cell Activation T-Cell Receptor T-Lymphocyte Technology Testing Therapeutic Therapeutic Agents Time Toxic Shock Syndrome Toxic Shock Syndrome Toxin-1 Toxic effect Toxin Transgenic Mice Tumor Necrosis Factor-alpha Universities Work Yeasts aerosolized base crosslink cytokine experience human TNF protein immunoglobulin receptor in vitro activity in vivo member mouse model mutant receptor receptor binding response

项目摘要

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.

描述（由申请人提供）：细菌肠毒素是一类被称为超抗原（SAg）的蛋白质的成员，其具有致死活性，因为它们引起大量T细胞活化，导致释放炎症分子，如TNF-α。大多数已知的细菌SAg由金黄色葡萄球菌和化脓性链球菌表达。这些包括生物恐怖主义的选择剂，如葡萄球菌肠毒素B（SE B），美国军方认为这是丧失能力和致命性的主要威胁。SAg通过将T细胞受体V β区域与另一细胞上的II类MHC分子交联来激活T细胞。该项目将开发可溶性Vbeta受体，可以中和肠毒素，并将涉及四位科学家的实验室，他们在SAgs的结构，功能和临床研究方面具有丰富的经验：大卫克兰兹，伊利诺伊大学;帕特里克施利弗特，美国。明尼苏达州;马里兰州;西娜·巴瓦里，美国陆军。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：SEB，TSST-1，SEC 3，SpeA，SpeC）。基于预测的SEB对人体的LD 50值较低（约1 μ g），很可能需要低皮摩尔亲和力试剂来有效中和。在以前的工作中，我们雪，高亲和力，可溶性受体结构域（12 kDa）的酵母展示工程抑制SEB和SEC 3的体外活性。作为Vbeta：SAg相互作用可被工程化至低皮摩尔亲和力的证据，我们最近产生了一组Vbeta 8突变体，其对SEB的KD值为40至90 pM。本项目将进一步开发这些和其他可溶性Va受体作为治疗剂。具体目标是：1）工程化和表征以高亲和力结合SAg的Vbeta区：SEB、TSST-1、SEC 3、SpeA和SpeC，2）产生来自目的1的试剂的Vbeta免疫球蛋白融合体并表征其在小鼠中的结合特性和血清寿命，3）测试可溶性Vbeta-Ig融合体在体外抑制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”II类MHC/CD 4转基因小鼠（包括暴露的气溶胶模型）和兔子，它们在症状和毒性方面类似于人类SAg介导的疾病。这里开发的策略工程非常高的亲和力中和受体，并增加其血清寿命应直接转化为开发拮抗剂对其他毒素被认为是潜在的生物恐怖主义代理。