Infection Site Targeted Antitoxin Antibody (ISTAb) against Bacillus anthracis

项目摘要 革兰氏阳性细菌炭疽芽孢杆菌是潜在的生物武器化的非常有力的候选者，以及 据信实际上已被前苏联武器化。炭疽孢子很容易在 天然或在实验室中生产的，能抵抗恶劣条件，并能在 环境。微小的孢子可以制成粉末、喷雾剂、食物或水的形式。两种关键毒素 由保护性抗原(PA)与致死因子(LF)或水肿性因子(EF)结合而产生 炭疽杆菌毒力的关键作用。美国疾病控制与预防中心在可能暴露于 炭疽杆菌气雾化孢子由口服抗生素和以PA为基础的炭疽疫苗组成。 然而，在实践中，这些治疗方法不能充分解决细菌毒素的不良影响。 发布曝光后的照片。在这个R41提案中，我们打算开发一种新的方法来针对中和反 针对感染部位的毒素抗体。该方法利用细胞壁靶向结构域(CWT) 特性良好的噬菌体内毒素：PlyG、PlyL和PlyB，它们具有物种特异性和高亲和力 对炭疽芽胞杆菌细胞壁成分的影响。这些CWTs将与特定的抗毒素中和单抗融合 产生感染部位靶向抗毒素抗体(ISTAbs)的抗体。预计ISTAb将 在最需要它们的感染部位蓄积，并捕获和隔离毒素，从而 立即中和毒素的影响，防止它们释放到循环中。细菌毒素 然后，复合体有望被吞噬细胞清除。在这项提案中，我们将使用三种炭疽-PA中和 将单抗与高亲和力噬菌体内溶蛋白CWTS融合，产生ISTAb。在《目标1》中，我们将放映 获得来自10种噬菌体内毒素的最佳结合CWTs，包括来自PlyG、PlyL和PlyB的内毒素。我们会 根据体内和体外结合来表征它们。在目标2中，根据目标1的结果，我们将选择3个CWTS 通过将CWTS与三个高度中和的抗炭疽单抗融合而产生多达九个ISTAb 以抗体为支架，对其体外结合活性和毒素中和活性进行表征。在《目标3》中，我们将 根据稳定性、细菌细胞结合的特异性和亲和力进一步表征所选的ISTAb，以及 吞噬细胞杀伤试验中的表现。在目标4中，我们将在挑战前和 激发治疗后的小鼠模型，并探索ISTAbs的潜在免疫原性。 由于ISTAb技术提供了两个治疗优势：立即中和毒素 感染和吞噬细胞对吞噬细胞的杀伤，这些分子很可能会 与现有的抗生素协同作用。立即清除毒素、吞噬杀灭和 同时使用抗生素有望产生协同效应，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，这项技术还可以应用于各种其他细菌 毒素在致病机制中起关键作用的病原体。总体而言，这种方法在董事会中得到了应用 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.