Infection Site Targeted Antitoxin Antibody (ISTAb) against Bacillus anthracis

项目摘要 革兰氏阳性细菌炭疽杆菌是潜在生物武器的一个非常强的候选者， 被认为是前苏联制造的武器炭疽孢子很容易在 自然或在实验室中产生的，能抵抗恶劣的条件，并能在环境中存活很长时间。 环境这种微小的孢子可以制成粉末、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）联合作用产生 在B中的关键作用。炭疽毒力潜在暴露后的CDC建议 雾化的B。炭疽孢子由口服抗生素和PA基炭疽疫苗的组合组成。 然而，在实践中，这些治疗不能充分解决细菌毒素的不良影响 曝光后释放。在这项R41提案中，我们打算开发一种新的方法来靶向中和抗- 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） 充分表征的噬菌体内溶素：PlyG、PlyL和PlyB，其以物种特异性和高亲和力结合 到B的细胞壁组分。炭疽病这些CWT将与特异性抗毒素中和单克隆抗体融合， 抗体以产生感染位点靶向抗毒素抗体（ISTAb）。预计ISAB将 在最需要它们的感染部位积累，并捕获和隔离毒素， 立即中和毒素的影响并防止其释放到循环中。细菌毒素 然后预期复合物被吞噬细胞清除。在这个建议中，我们将使用三种炭疽-PA中和 融合至高亲和力噬菌体内溶素CWT的单克隆抗体产生ISTAb。在目标1中， 对于来自十种噬菌体内溶素的最佳结合CWT，包括来自PlyG、PlyL和PlyB的那些。我们将 基于体内和体外结合来表征它们。在目标2中，基于目标1的结果，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， 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， 基于稳定性、细菌细胞结合特异性和亲和力进一步表征所选ISTAb，和 在调理吞噬细胞杀伤测定中的性能。在目标4中，我们将在攻毒前进行有效性检测， 攻击后处理小鼠模型，并且还探索ISTAb的潜在免疫原性。 由于ISAB技术提供了两个治疗优势：在感染部位立即中和毒素， 感染和吞噬细胞的调理吞噬杀伤，这些分子很有可能 与现有抗生素协同作用。立即清除毒素，吞噬细胞杀伤， 同时使用抗生素有望产生协同作用，并产生远优于目前的上级治疗。 疫苗加抗生素的标准。此外，该技术还可以应用于多种其他细菌。 毒素在致病过程中起关键作用的病原体。总的来说，这种方法具有广泛的应用性， 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.