Bifunctional antibodies with targeted CNS delivery against West Nile Virus

项目摘要/摘要 西尼罗河病毒(WNV)可引起多种脊椎动物中枢神经系统的感染 物种。感染西尼罗河病毒的人会患上脑膜炎和脑炎，老年人和 免疫功能受损的人患严重神经系统疾病和死亡的风险最大。西尼罗河病毒的新威胁 在全球范围内，由于缺乏可用的治疗方法，需要研究开发有效的治疗方法和 能够以较低的成本将候选疗法快速转移到临床护理环境中的生产技术- 有意识的举止。我们最近开发了一种植物来源的人源化单抗，具有很好的治疗效果。 潜在的，与所需的人类N-连接糖基化模式。该单抗(HE16)与高度保守的 全世界几乎所有分离物的包膜蛋白上的表位，并显示出很有希望的暴露后 治疗活动。尽管如此，我们的研究表明，外周注射hE16的时间窗口有限 对啮齿动物的疗效：通过静脉或腹膜途径单次注射hE16 在感染后第5天或更早，可提高存活率。相比之下，将hE16直接导入 感染后第6天的大脑增强了对致死性西尼罗河病毒感染的保护作用。我们的预赛 数据显示，一种基因工程的hE16双功能单抗变体(TFR-Bif)具有潜在的 血脑屏障(BBB)的增强穿透可在CHO细胞或植物中表达。TFR-BIF保留 其结合和中和西尼罗河病毒的能力，但重要的是获得了结合转铁蛋白受体(TFR)和 内吞进入小鼠脑内皮细胞。在这笔赠款的R21阶段，我们将使用小鼠TFR- 在西尼罗河病毒感染的小鼠模型中，特异性TFR-Bif作为原则证明。我们将检验这一假设 TFR-BIF可达到较高的中枢神经系统水平，并延长了治疗西尼罗河病毒的窗口 脑炎。为了解决大脑中的关键安全问题，我们还将确定TFR-Bif糖类提供 全双功能与有限或特定的Fc受体(Fc？R)或C1q结合以消除潜在的抗体- 病毒感染或致病性炎症的依赖性增强(ADE)。在R33阶段，我们将 开发一种具有人类治疗潜力的类似双功能单抗，并测试植物是否提供 在安全性、生产成本和可伸缩性方面具有独特的优势，适用于cGMP下的大规模生产。在……里面 除了为西尼罗河病毒产生新的治疗试剂外，这项合作研究还将提供一个平台 用于向中枢神经系统输送单抗，这应该适用于其他感染性疾病的治疗， 中枢神经系统的炎症性或肿瘤性疾病。

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.