Infection Site Targeted Antitoxin Antibody (ISTAb) against Bacillus anthracis

项目概要 革兰氏阳性细菌炭疽杆菌是潜在生物武器化的有力候选者，并且 据信实际上已被前苏联武器化。炭疽孢子很容易发现于 自然或实验室生产，能抵抗恶劣的条件，可以在环境中长期生存 环境。微观孢子可以配制为粉末、喷雾剂、食物或水。两种关键毒素 由保护性抗原 (PA) 与致死因子 (LF) 或水肿因子 (EF) 结合产生 在炭疽芽孢杆菌毒力中起关键作用。潜在接触后 CDC 的当前建议 雾化炭疽芽孢杆菌孢子由口服抗生素和基于 PA 的炭疽疫苗组合而成。 然而，在实践中，这些治疗方法并不能充分解决细菌毒素的不利影响 曝光后发布。在这个 R41 提案中，我们打算开发一种新方法来中和抗- 特异性针对感染部位的毒素抗体。该方法利用细胞壁靶向域 (CWT) 充分表征的噬菌体内溶素：PlyG、PlyL 和 PlyB，它们以物种特异性和高亲和力结合 炭疽芽孢杆菌的细胞壁成分。这些 CWT 将与特定的抗毒素中和单克隆抗体融合 抗体产生感染部位靶向抗毒素抗体（ISTAb）。 ISTAB 预计将 在最需要的感染部位积聚，捕获并隔离毒素，从而 立即中和毒素的影响并防止其释放到循环中。细菌毒素 然后预计复合物将被吞噬细胞清除。在本提案中，我们将使用三种炭疽-PA 中和剂 单克隆抗体与高亲和力噬菌体内溶素 CWT 融合以生成 ISTAb。在目标 1 中，我们将筛选 十种噬菌体内溶素（包括来自 PlyG、PlyL 和 PlyB 的噬菌体内溶素）的最佳结合 CWT。我们会 根据体内和体外结合来表征它们。在目标 2 中，基于目标 1 结果，我们将选择 3 个 CWT 通过将 CWT 与三种高度中和的抗炭疽单克隆抗体融合，生成多达 9 种 ISTAb 抗体作为支架并表征它们的体外结合和毒素中和活性。在目标 3 中，我们将 根据稳定性进一步表征所选的 ISTAb；细菌细胞结合特异性和亲和力，以及 调理吞噬杀伤试验中的性能。在目标 4 中，我们将在攻击前和攻击前进行功效测试 攻击后治疗小鼠模型，并探索 ISTAb 的潜在免疫原性。 由于 ISTAb 技术提供了两个治疗优势： 感染和吞噬细胞调理吞噬杀伤，这些分子很可能会 与现有抗生素有协同作用。立即清除毒素、吞噬细胞杀灭和 同时使用抗生素有望产生协同作用并产生远远优于目前的治疗方法 疫苗加抗生素的标准。此外，该技术还可应用于多种其他细菌 毒素在发病机制中起关键作用的病原体。总的来说，这种方法在电路板应用中作为一种 跨多种病原体的平台技术。

Project Summary The Gram-positive bacterium Bacillus anthracis is a very strong candidate for potential bio- weaponization, and believed to have actually been weaponized by the former Soviet Union. Anthrax spores are readily found in nature or produced in the laboratory, are resistant to harsh conditions, and can survive for a long time in the environment. The microscopic spores could be formulated in powder form, sprays, food, or water. Two key toxins generated by combination of the protective antigen (PA) with either lethal factor (LF) or edema factor (EF) play a critical role in B. anthracis virulence. Current CDC recommendations following potential exposure to aerosolized B. anthracis spores consist of a combination of oral antibiotics and PA-based anthrax vaccine. However, in practice, these treatments cannot adequately address the adverse effects of bacterial toxins released post exposure. In this R41 proposal we intend to develop a novel approach to target neutralizing anti- toxin antibodies specifically to the site of infection. The approach exploits the cell wall targeting domains (CWT) of well characterized phage endolysins: PlyG, PlyL and PlyB which bind with species-specificity and high affinity to cell wall components of B. anthracis. Theses CWTs will be fused to specific antitoxin neutralizing monoclonal antibodies to generate Infection Site Targeted Antitoxin antibodies (ISTAbs). ISTAbs are expected to accumulate at the site of infection where they are needed most, and capture and sequester the toxins, thus immediately neutralizing the effects of the toxins and preventing their release into circulation. Bacterium-toxin complex is then expected to be cleared by phagocytes. In this proposal, we will use three anthrax-PA neutralizing monoclonal antibodies fused to high affinity phage endolysin CWTs to generate ISTAbs. In Aim 1 we will screen for best binding CWTs from ten phage endolysins, including those from PlyG, PlyL, and PlyB. We will characterize them based on in vivo and in vitro binding. In Aim 2, based on Aim 1 results, we will select 3 CWTs for generating up to nine ISTAbs by fusing the CWTs with three highly neutralizing anti-Anthrax monoclonal antibody as scaffold and characterize them for in vitro binding and toxin neutralizing activity. In Aim 3 we will further characterize the selected ISTAbs based on stability; bacterial cell binding specificity and affinity, and performance in opsonophagocytic killing assays. In Aim 4, we will perform efficacy testing in pre-challenge and post challenge treatment mouse models and also explore potential immunogenicity of the ISTAbs. Since ISTAb technology provides two therapeutic advantages: immediate toxin neutralization at the site of infection and opsonophagocytic killing by phagocyte, there is a high probability that these molecules will synergize with existing antibiotics. The combination of immediate toxin clearance, phagocytic killing, and concurrent use of antibiotics is expected to create synergy and yield a treatment that is far superior to the current standard of vaccine plus antibiotics. Furthermore, this technology can be applied to a variety of other bacterial pathogens where toxins play a key role in pathogenesis. Overall, this approach has board application as a platform technology across multiple pathogens.