Novel immunotherapeutics against multidrug-resistant Neisseria gonorrhoea

摘要 2013年，疾控中心列出的淋病奈瑟菌(Ng)被列为全球对人类健康构成紧急威胁的三种多重耐药病原体之一。NG已经对几乎所有的抗菌药产生了抗药性，并达到了“超级细菌”的状态。淋病的并发症包括盆腔炎，这可能导致不孕和异位妊娠。迫切需要针对这种病原体的新的治疗方法。产业界和学术界的这一合作旨在进一步开发两种针对耐药Ng的免疫疗法。每个分子都针对Ng上的一个不同的表位；两者在发病机制中都是重要的，因此在体内普遍表达。靶向对毒力具有重要作用的不同表位的优点包括：1)协同活性；2)如果发生耐药，则增加了发展的障碍。嵌合单抗靶向Ng并在体内介导补体(C‘)依赖的对Ng的杀伤。包括Ng在内的几种病原体通过与称为因子H(FH)的宿主C‘抑制物结合来逃避C’。FH由20个域组成，以扩展的首尾方式排列。FH区18-20(缺乏C‘抑制功能)与Ig G Fc融合，可产生C’依赖性杀伤Ng的作用。FH结构域19和20对于限制宿主组织上不想要的C‘激活很重要。我们在FH18-20/Fc的19区引入了D-→G氨基酸突变。这种被称为FH*/Fc的蛋白质在体外和体内都保持了对Ng的效力，但不能裂解宿主细胞。在Aim 1 Planet Biotech，Inc.将在烟草植物中生产嵌合mAb和FH*/Fc。这将允许高效和低成本的生产。植物产生的FH*/Fc和抗Ng的嵌合mAb将在体外和小鼠中由UMass2进行测试。这些药物将被给予：i)系统地(模拟男性和妇女已建立的感染的辅助治疗)或ii)阴道内(模拟高危妇女的局部免疫预防)。每个分子都将单独和联合进行过敏测试，以提高抗药性的屏障。在表达人类C‘抑制物FH和C4b结合蛋白的新型转基因(TG)小鼠中也将评估疗效，Ng在人类特有的人身上结合以逃避C’，因此代表着对人类可能遇到的药物的障碍。在目标3中，橡树冠研究所将把这两种药物配制成缓释阴道环，用作局部免疫预防。在AIM 4中，Xenomettics将在大鼠和食蟹猴身上进行毒理学和毒代动力学(TK)研究，而比较生物科学将进行组织交叉反应研究。在目的5中，将利用缺乏C3、C5、PMN和/或巨噬细胞的小鼠在体内定义嵌合mAb和FH*/Fc的作用机制，以确定体内保护的相关性。这些研究可能导致准备和参加与FDA的IND前会议(AIM 6)。

ABSTRACT In 2013, the CDC listed Neisseria gonorrhoeae (Ng) is listed as one of the three multidrug-resistant pathogens that represented ‘urgent’ threats to human health worldwide. Ng has become resistant to almost every antibi-otic and has achieved “superbug” status. Complications of gonorrhea include pelvic inflammatory disease, which may lead to infertility and ectopic pregnancy. Novel therapeutics against this pathogen are urgently needed. This collaboration between industry and academia seeks to further preclinical development of two immunotherapeutics against drug-resistant Ng. Each molecule targets a distinct epitope on Ng; both are im-portant for pathogenesis and thus ubiquitously expressed in vivo. Advantages of targeting distinct epitopes im-portant for virulence include i) synergistic activity and ii) raising the barrier for the development of drug re-sistance, if it were to occur. A chimeric mAb targets Ng and mediates complement (C′)-dependent killing of Ng in vivo. Several pathogens including Ng evade C′ by binding to a host C′ inhibitor called factor H (FH). FH comprises 20 domains, arranged in an extended head-to-tail fashion. FH domains 18-20 (lacks C′ inhibitory function) fused to IgG Fc effects C′-dependent killing of Ng. FH domains 19 and 20 are important to limit un-wanted C′ activation on host tissue. We introduced a D→G amino acid point mutation in domain 19 of FH18-20/Fc. This protein, called FH*/Fc, retains its efficacy against Ng in vitro and in vivo but does not lyse host cells. In Aim 1 Planet Biotechnology, Inc. will produce the chimeric mAb and FH*/Fc in tobacco plants. This will permit efficient and low cost production. The efficacy of plant-produced FH*/Fc and the chimeric mAb against Ng in vitro and in mice will be tested by UMass in Aim 2. The drugs will be administered: i) systemically (mod-eling adjunctive treatment of established infection in men and women) or ii) intravaginally (modeling topical immunoprophylaxis in high-risk women). Each molecule will be tested individually and in combination for syn-ergy and to raise the barrier for drug resistance. Efficacy will also be assessed in novel transgenic (Tg) mice that express the human C′ inhibitors, FH and C4b-binding protein, which Ng bind to in a human-specific man-ner to evade C′, and therefore represent barriers to the drugs that may be encountered in humans. In Aim 3, Oak Crest Institute will formulate both drugs in combination in slow-release vaginal rings for use as topical im-munoprophylactics. In Aim 4 Xenometrics will perform toxicology and toxicokinetic (TK) studies in rats and cynomolgus monkeys and Comparative Biosciences will perform tissue-cross reactivity studies. In Aim 5, the mechanism of action of the chimeric mAb and FH*/Fc and will be defined in vivo using mice that lack C3, C5, PMNs and/or macrophages to define correlates of protection in vivo. These studies may lead to preparation for and participation in a pre-IND meeting with FDA (Aim 6).