Antibody Targeting of KPC and NDM1

描述（由申请人提供）：在过去的十年中，多重耐药生物（mdro）已经成为世界各地卫生保健系统的地方性疾病。新的治疗策略最优先考虑的是由极端耐药（XDR）革兰氏阴性杆菌（GNB）引起的感染，这种细菌对碳青霉烯类和除粘菌素或替加环素以外的所有其他抗生素具有耐药性。例如，由XDR引起的感染，表达kpc的肺炎克雷伯菌已经在美国蔓延，并与50%的死亡率相关。与KPC一样，NDM-1耐药机制通过可传播的质粒传播，并已在世界范围内发现，包括亚洲、西欧和美国。迫切需要预防或治疗这些广泛耐药细菌引起的感染的新方法，特别是考虑到治疗这些细菌的抗生素缺乏。处理表达KPC和NDM-1碳青霉烯酶的病原体的一种新的潜在方法是中和酶的活性和/或靶向表达它们的细菌进行破坏。用重组KPC-1 （rKPC1）或NDM-1 （rNDM1）免疫产生抗KPC-1和NDM-1的多克隆抗体。当纯化的IgG与铁铁载体共价连接以增强细菌摄取时，2.5¿g/ml的复合免疫而非控制的IgG铁载体杀死表达ndm -1的肺炎克雷伯菌。铁载体与免疫但不控制的IgG Fabc片段结合，通过减少IgG重链铰链区域的二硫键形成，可以杀死表达NDM-1和kpc的菌株。IgG杀死细菌的机制尚不清楚。据推测，与铁载体的结合可以通过增加抗体进入质周间隙或通过抗体介导的铁载体摄取通道的阻塞来增加杀伤活性，从而阻断生长所需的铁摄取。因此，本研究的具体目标是：1)明确抗体摄取和铁在体外杀死抗NDM-1和KPC-1的铁载体偶联多克隆IgG和Fabc中的作用；2)通过工程单克隆抗体（mab）靶向一致的KPC和NDM酶，并将其偶联到铁载体上，从而优化效果。拟议的研究将确定一种创新的、有前途的技术的机制，以攻击高度耐药的GNB。通过铁载体增强单抗摄取特别有希望，因为许多抗生素耐药靶点存在于质周空间，这也是铁载体在GNB中沉积铁的直接位置。因此，目前提案的结果对增强针对一般GNB的基于抗体的治疗具有根本意义，并有可能实现攻击XDR病原体的全新方法。目前的结果将支持未来的R01和/或SBIR/STTR应用，以人源化单克隆抗体，确定其在体内治疗多种XDR GNB菌株和物种的潜力，并确定生物化学杀伤机制。

DESCRIPTION (provided by applicant): In the last decade, multi-drug resistant organisms (MDROs) have become endemic in healthcare systems throughout the world. Of highest priority for new treatment strategies are infections caused by extreme drug resistant (XDR) Gram negative bacilli (GNB), which are resistant to carbapenems and all other antibiotics except for colistin or tigecycline. For example, infections caused by XDR, KPC-expressing Klebsiella pneumoniae have spread across the US and are associated with a 50% mortality rate. Like KPC, the NDM-1 resistance mechanism is spread by transmissible plasmids, and has been found worldwide, including in, Asia, Western Europe and the US. New ways to prevent or treat infections caused by these XDR bacteria are critically needed, especially given the dearth of antibiotics in the pipeline to treat them. A novel potential means to deal with pathogens expressing KPC and NDM-1 carbapenemases is to neutralize the activity of the enzymes and/or target bacteria expressing them for destruction. Polyclonal anti- KPC-1 and NDM-1 antibodies were raised by immunization with recombinant KPC-1 (rKPC1) or NDM-1 (rNDM1). When purified IgG was covalently linked to an iron siderophore to enhance bacterial uptake, 2.5 ¿g/ml of the complexed immune but not control IgG-siderophore killed NDM-1-expressing K. pneumoniae. Siderophore conjugation to immune but not control IgG Fabc fragments, formed by reducing the disulfide bonds at the IgG heavy chain hinge region, enabled killing of both NDM-1- and KPC-expressing strains. The mechanisms by which the IgG kill the bacteria is unclear. It is hypothesized that conjugation to the siderophore increases cidal activity either by increasing antibody uptake into the periplasmic space, or by antibody-mediated obstruction of the siderophore uptake channel, thereby blocking iron uptake needed for growth. Thus, the specific aims are to 1) Define the role of antibody uptake and iron in in vitro killing of siderophore-conjugated polyclonal IgG and Fabc raised against NDM-1 and KPC-1 and 2) Optimize efficacy by engineering monoclonal antibodies (MAbs) targeting consensus KPC and NDM enzymes and conjugating them to iron siderophores or not. The proposed investigations will define the mechanisms of an innovative, promising technology to attack highly resistant GNB. Enhanced MAb uptake via siderophores is particularly promising because many antibiotic resistance targets exist in the periplasmic space, which is also the immediate location siderophores deposit iron in GNB. Hence, results from the current proposal have fundamental implications for enhancing antibody-based therapeutics against GNB in general, and have the potential to enable an entirely new approach to attacking XDR pathogens. The current results will support a future R01 and/or SBIR/STTR application to humanize the MAbs, define their potential to treat multiple strains and species of XDR GNB in vivo, and define biochemical mechanisms of killing.