ISTAb- A novel therapy to target staphylococcal toxins at the site of infections

项目摘要 许多细菌病原体分泌外毒素以改变宿主-病原体的相互作用，从而有利于 细菌。有几个成功的通过靶向细菌毒素进行预防和治疗的例子 破伤风、白喉和百日咳疫苗数十年的历史证明了这一点。金黄色葡萄球菌 (沙门氏菌)是一种主要的人类病原体，导致数十万人住院和12,000多人住院 美国每年都有人死亡。SA产生过多的毒素，包括毛孔形成毒素(PFTs)，它们发挥着 在发病机制和免疫逃避中起关键作用。同时用单抗中和这些毒素 单抗有望减少临床疾病，但不太可能根除侵袭性疾病，最近的研究表明 PFT单抗鸡尾酒未能预防SA呼吸机相关性肺炎(Arsanis临床试验)。我们有 设计了一种新的方法，针对感染部位的特异性中和抗毒素单抗，使即时 毒素中和与介导性吞噬细胞杀伤(OPK)同时进行。该方法利用了 噬菌体来源的溶菌素的细胞壁靶向结构域(CWT) 对特定细菌细胞壁成分的亲和力。将CWT与特定的抗毒素单抗融合，产生 感染部位靶向抗毒素抗体(ISTAbs)。我们已经成功地将其应用于炭疽杆菌和C. NIAID支持下的艰难梭菌。在这项建议中，我们的目标是利用分离的CWT开发金黄色葡萄球菌的ISTAb。 溶葡萄球菌酶和有效的PFT中和单抗。ISTAb预计会在它们所在的地方积累起来 最需要的，即在感染部位；它们捕获和隔离毒素，从而立即中和 毒素的免疫抑制作用和防止其释放到循环中。细菌-ISTAb-毒素 然后，复合体被吞噬细胞清除。同时进行毒素中和和细菌清除是独一无二的 ISTAb技术相对于单纯的抗毒素治疗的优势。 在这项建议中，我们将使用一组广泛中和的金黄色葡萄球菌PFT单抗来生成ISTAb 溶葡萄球菌酶CWT。然而，由于免疫球蛋白G与金黄色葡萄球菌表面的蛋白A结合会干扰 吞噬细胞杀伤，甚至可能导致免疫病理，我们将创建和测试突变的ISTAbs 不结合蛋白A。在目标1中，将产生这些ISTab并对其进行生物物理和功能鉴定。 特性，包括中和和OPK活性，以选择用于体内疗效研究的短名单。在目标2中， 我们将评估ISTAbs在不同金黄色葡萄球菌感染小鼠模型中的疗效，包括肺炎， 脓毒症和手术伤口感染。在第一阶段SBIR完成后，我们预计第二阶段项目将 将疗效研究扩展到兔侵袭性感染模型，包括脓毒症、人工关节感染、 呼吸机相关性肺炎和心内膜炎模型以及IND使能研究。

Project Summary Many bacterial pathogens secrete exotoxins to modify the host-pathogen interactions in a manner that benefits the bacteria. There are several examples of successful prophylaxis and treatment by targeting bacterial toxins as evident by decades-long history of vaccines for tetanus, diphtheria, and pertussis. Staphylococcus aureus (SA) is a major human pathogen responsible for several hundred thousand of hospitalizations and over 12,000 death in the US every year. SA produces a plethora of toxins including pore-forming toxins (PFTs) that play a key role in pathogenesis and immune evasion. While neutralization of these toxins by monoclonal antibodies (mAbs) is expected to reduce clinical disease, it is unlikely to uproot invasive disease, as suggested by recent failure of a PFT mAb cocktail to prevent SA ventilator associated pneumonia (Arsanis clinical trial). We have devised a novel approach to target neutralizing anti-toxin mAbs specifically to the site of infection enabling instant toxin neutralization and mediating opsonophagocytic killing (OPK) at the same time. The approach exploits the cell wall targeting domains (CWT) of a phage-derived bacteriolysin which binds with species-specificity and high affinity to cell wall components of specific bacteria. The CWT is fused to specific anti-toxin mAbs to generate Infection Site Targeted Antitoxin antibodies (ISTAbs). We have successfully applied this to B. anthracis and C. difficile under NIAID support. In this proposal we aim to develop ISTAbs for S. aureus using the isolated CWT of lysostaphin along with potent PFT neutralizing mAbs. ISTAbs are expected to accumulate where they are needed most, i.e. at the site of infection; they capture and sequester the toxins, thus immediately neutralizing the immune suppressive effects of the toxins and preventing their release into circulation. Bacterium-ISTAb-toxin complex is then cleared by phagocytes. Concurrent toxin neutralization and bacterial clearance is a unique advantage of the ISTAb technology over mere antitoxin treatment. In this proposal, we will use a set of broadly neutralizing S. aureus PFT mAbs to generate ISTAbs with lysostaphin CWT. However, because IgG binding to protein A on the surface of S. aureus interferes with opsonophagocytic killing and may even cause immunopathology, we will create and test mutant ISTAbs that do not bind protein A. In Aim 1, these ISTAbs will be generated and characterized for biophysical and functional properties, including neutralization and OPK activity, to select a short list for in vivo efficacy studies. In Aim 2, we will evaluate the efficacy of ISTAbs in different mouse models of S. aureus infection including pneumonia, sepsis and surgical wound infection. Upon completion of the Phase I SBIR we anticipate a Phase II project to expand the efficacy studies to invasive infection models in rabbits including sepsis, prosthetic joint infection, ventilator associated pneumonia, and endocarditis models as well as IND-enabling studies.