Infection Site Targeted Antitoxin Antibody (ISTAb) against Bacillus anthracis

项目摘要 炭疽芽孢杆菌是一种革兰氏阳性芽胞形成菌，被列为最高致病菌。 NIAID和CDC的关注(A类)。BA易于生长，其孢子可配制成高密度的 形成稳定的粉末并以气雾剂的形式传播，或用于污染食物或水。2001年，字母加了花边 用炭疽菌粉将孢子邮寄给几位美国政客。22个人，包括12封邮件 都被感染了，其中五人死亡。炭疽杆菌的毒力在很大程度上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)组合而成 因数(EF)。虽然一些口服抗生素和疫苗可以使用，但在实践中，这些治疗方法 不能充分解决暴露后释放的细菌毒素的不良影响。在我们最近 完成R41项目，我们开发并测试了一种靶向中和抗PA抗体的新方法 特别是在体外和体内的感染部位。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)与物种特异性和 对细胞壁组分具有较高的亲和力。这些CWTs被融合到特定的抗毒素中和 产生感染部位靶向抗毒素抗体(ISTAbs)的单抗。ISTAb 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，并通过吞噬细胞杀死吞噬细胞，同时清除细菌和 毒素。我们比较了9个ISTAb候选者(3个CWTs和3个mAbs)基于体外分析(细胞 结合和毒素中和)，并选择了一种ISTAb(AVP-21D9-PlyG)用于攻击前后 在老鼠身上进行的活体研究。该ISTAb表现出明显高于亲本抗体的保护水平。这 R42的目标是将这种领先的ISTAb分子带入治疗流水线的下一个水平。在这份提案中， 我们将生产并广泛表征下一代AVP-21D9-PlyG ISTAb，包括稳定性 并在小鼠和非人类灵长类动物(NHP)上进行体内疗效研究，并开发出稳定的配方。在……里面 目标1，我们将使用计算机辅助优化来生成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，两名领先的候选人将在 老鼠模型。一种铅分子将在NHP模型中进行PK和挑战后有效性测试。在……里面 目标3：最终的ISTAb将经历加速稳定性和PK研究、配方和生成 在CHO-S细胞中获得稳定的细胞系。立即清除毒素、吞噬杀灭和 同时使用抗生素，预计将产生协同效应，并产生远远优于 目前的护理标准。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 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.