Bifunctional antibodies with targeted CNS delivery against West Nile Virus

项目总结/摘要 西尼罗河病毒（West Nile Virus，WNV）是一种可引起多种脊椎动物中枢神经系统感染的病毒 物种感染西尼罗河病毒的人可发展为脑膜炎和脑炎，老年人和 免疫功能低下者患严重神经系统疾病和死亡风险最大。西尼罗河病毒的新威胁 在全球范围内，缺乏可用的治疗方法，需要研究开发有效的治疗方法， 生产技术，可以迅速转移候选疗法到临床护理设置的成本， 有意识的方式。我们最近开发了一种植物来源的人源化单克隆抗体，具有很好的治疗作用。 潜在的，具有所需的人N-连接糖基化模式。该单克隆抗体（hE 16）结合高度保守的 表位上的包膜蛋白，几乎所有的世界各地的分离株，并显示出有前途的暴露后 治疗活性。尽管如此，我们的研究表明hE 16的外周递送具有有限的时间窗， 在啮齿类动物中的功效：通过静脉内或腹膜内途径施用单剂量的hE 16 在感染后第5天或更早时，提高存活率。相比之下，将hE 16直接递送到 在感染后第6天，仓鼠的大脑改善了对致死性WNV感染的保护。我们的初步 数据显示，一种基因工程改造的hE 16双功能单克隆抗体变体（TfR-Bif）具有潜在的 增强的血脑屏障（BBB）穿越可以在CHO细胞或植物中表达。TfR-Bif保留 其结合和中和WNV的能力，但重要的是获得结合转铁蛋白受体（TfR）的能力， 内吞进入小鼠脑内皮细胞。在R21阶段，我们将使用小鼠TfR- 特异性TfR-Bif作为WNV感染小鼠模型中的原理验证。我们将检验这个假设， TfR-Bif可以在CNS中达到更高的水平，并扩展了针对WNV的治疗窗口 脑炎为了解决大脑中的关键安全问题，我们还将鉴定TfR-Bif糖型， 具有有限或特异性Fc受体（Fc R）或C1 q结合的完全双功能性，以消除潜在的抗体- 病毒感染或致病性炎症的依赖性增强（ADE）。在R33阶段， 开发具有人类治疗潜力的类似双功能mAb，并测试植物是否提供 在安全性、生产成本和cGMP下大规模生产的可扩展性方面具有独特的优势。在 除了产生新的治疗西尼罗河病毒的试剂，这项合作研究将提供一个平台， 用于将MAb递送至CNS，这应该适用于治疗其他感染性疾病， 炎性或肿瘤性疾病。

PROJECT SUMMARY/ABSTRACT West Nile Virus (WNV) causes infection in the central nervous system (CNS) in several vertebrate animal species. Humans infected with WNV can develop meningitis and encephalitis, and the elderly and immunocompromised are at greatest risk for severe neurological disease and death. New threats of WNV globally and the lack of available treatments warrant studies to develop effective therapeutics and production technologies that can rapidly transfer candidate therapies into the clinical care setting in a cost- conscious manner. We recently developed a plant-derived humanized MAb with promising therapeutic potential, with a desired human N-linked glycosylation pattern. This MAb (hE16) binds to a highly conserved epitope on the envelope protein of virtually all isolates worldwide and shows promising post-exposure therapeutic activity. Nonetheless, our studies show that peripheral delivery of hE16 has a limited window of efficacy in rodents: administration of a single dose of hE16 through an intravenous or intraperitoneal route at day 5 post infection or earlier improves survival rates. In comparison, delivery of hE16 directly into the brain at day 6 after infection improved protection against lethal WNV infection in hamsters. Our preliminary data show that a genetically engineered bifunctional MAb variant of hE16 (TfR-Bif) with the potential for enhanced crossing of the blood-brain barrier (BBB) can be expressed in CHO cells or plants. TfR-Bif retains its ability to bind and neutralize WNV, but importantly gains the ability to bind transferrin receptor (TfR) and endocytose into mouse brain endothelial cells. In the R21 phase of this grant, we will use the mouse TfR- specific TfR-Bif as a proof-of-principle in a mouse model of WNV infection. We will test the hypothesis that TfR-Bif can achieve higher levels in the CNS and extends the window of treatment against WNV encephalitis. To address critical safety issues in the brain, we will also identify TfR-Bif glycoforms that offer full bifunctionality with limited or specific Fc receptor (Fc¿R) or C1q binding to eliminate potential antibody- dependent enhancement (ADE) of virus infection or pathogenic inflammation. In the R33 phase, we will develop an analogous bifunctional mAb with human therapeutic potential and test if plants provide distinctive advantages in safety, production cost, and scalability for large-scale production under cGMP. In addition to generating novel therapeutic reagents for WNV, this collaborative study will provides a platform for delivery of MAbs to the CNS, which should be applicable to the treatment of other infectious, inflammatory, or neoplastic diseases in the CNS.