Infection Site Targeted Antitoxin Antibody (ISTAb) against Bacillus anthracis

项目摘要 炭疽芽孢杆菌（Ba）是一种革兰氏阳性孢子形成细菌，被列为最高的病原体。 关注（A类）由NIAID和CDC。BA易于生长，其孢子可以配制成高度浓缩的培养基。 形成稳定粉末，以气溶胶形式散布或用于污染食物或水。2001年， 寄给了几位美国政客22人，包括12封邮件 被感染，其中五人死亡B。炭疽的毒力主要取决于两种关键毒素 由与致死因子（LF）或水肿相关的保护性抗原（PA）结合产生 因子（EF）。虽然一些口服抗生素和疫苗可供使用，但在实践中， 不能充分解决暴露后释放的细菌毒素的不利影响。在我们最近 完成R41项目后，我们开发并测试了一种新的靶向中和抗PA抗体的方法 在体外和体内特异性地针对感染部位。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），其以种特异性结合， 对Ba细胞壁组分具有高亲和力。这些CWT融合到特定的抗毒素中和 在一些实施方案中，使用单克隆抗体（mAb）来产生感染位点靶向抗毒素抗体（ISTAb）。ISTA b 该技术提供了两个治疗优势：在感染部位立即中和毒素 防止毒血症，以及通过吞噬细胞的调理吞噬杀伤，以同时清除细菌和 毒素我们比较了九种ISTAb候选物（三种CWT和三种mAb），这些候选物基于体外测定（细胞培养）。 结合和毒素中和），并选择一种ISTAb（AVP-21 D9-PlyG）用于攻击前和攻击后， 小鼠体内研究。该ISAb表现出比亲本IgG显著更高的保护水平。这 R42旨在将这种领先的ISAB分子带入治疗管道的下一个水平。在这一提议中， 我们将生产和广泛表征下一代AVP-21 D9-PlyG ISTAb，包括稳定性 以及在小鼠和非人灵长类动物（NHP）中的体内功效研究，并开发稳定的制剂。在 目标1，我们将使用计算机辅助优化生成3-5个ISTAb变体，以消除潜在的 可能使下游发展复杂化的债务。在目标2中，两名主要候选人将在 小鼠模型。将在NHP模型中检测一种先导分子的PK和攻毒后疗效。在 目的3：将对最终ISAB进行加速稳定性和PK研究、配制和生成 CHO-S细胞中的稳定细胞系。立即清除毒素，吞噬细胞杀伤， 同时使用抗生素，预计将产生协同作用，并产生远远优于常规治疗的上级治疗。 目前的护理标准。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 Bacillus anthracis (Ba) is a Gram-positive spore forming bacterium that is listed as an agent of highest concern (Category A) by NIAID and CDC. Ba is easy to grow, and its spores can be formulated into highly stable powder form and disseminated as aerosol or used to contaminate food or water. In 2001, letters laced with powdered anthrax spores were mailed to several US politicians. Twenty-two people, including 12 mail handlers, were infected, and five of them died. B. anthracis virulence largely depends on two key toxins generated by combination of the protective antigen (PA) associated with either lethal factor (LF) or edema factor (EF). Although some oral antibiotics and a vaccine are available for use, in practice these treatments cannot adequately address the adverse effects of bacterial toxins released post exposure. In our recently completed R41 project, we developed and tested a novel approach to target neutralizing anti-PA antibodies specifically to the site of infection in vitro and in vivo. The approach exploits the cell wall targeting domains (CWT) of well characterized phage endolysins (PlyG, PlyL and PlyB) that bind with species-specificity and high affinity to cell wall components of Ba. These CWTs are fused to specific antitoxin neutralizing monoclonal antibodies (mAbs) to generate Infection Site Targeted Antitoxin antibodies (ISTAbs). ISTAb technology provides two therapeutic advantages: immediate toxin neutralization at the site of infection preventing toxemia, and opsonophagocytic killing by phagocytes to simultaneously clear both bacteria and toxin. We compared nine ISTAb candidates (three CWTs and three mAbs) based on in vitro assays (cell binding and toxin neutralization) and selected one ISTAb (AVP-21D9-PlyG) for pre- and post-challenge in vivo studies in mice. This ISTAb exhibited significantly higher level of protection than the parental IgG. This R42 is aimed to take this lead ISTAb molecule into the next level in therapeutic pipeline. In this proposal, we will produce and extensively characterize next-generation AVP-21D9-PlyG ISTAbs, including stability and in vivo efficacy studies in mice and nonhuman primates (NHP), and develop a stable formulation. In Aim 1, we will use computer-aided optimizations to generate 3-5 ISTAb variants to remove potential liabilities that may complicate downstream development. In Aim 2, two lead candidates will be tested in mouse models. One lead molecule will be tested in an NHP model for PK and post-challenge efficacy. In Aim 3: The final ISTAb will be subjected to accelerated stability and PK studies, formulation, and generation of stable cell lines in CHO-S cells. 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 care. 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.