Therapeutic Monoclonal Antibodies for Human Prion Diseases

描述（由申请人提供）：本拨款申请的主要研究假设是抗朊病毒单克隆抗体（mab）被动免疫可有效预防外中枢神经系统朊病毒感染引起的神经系统后果。朊病毒疾病（prionses）是一种传染性的、致命的神经退行性疾病，与细胞朊病毒蛋白PrPC转化为毒性、传染性和自我复制的PrPSC构象有关，目前尚无有效的治疗方法。由于输血或器官移植、摄入受朊病毒污染的食物或使用受朊病毒污染的手术器械，可能会发生人类意外的朊病毒感染。在中枢神经系统外暴露后，PrPSc在中枢神经系统侵入引起临床症状之前在淋巴器官内复制数月至数年。因此，在前中枢神经系统阶段的朊病毒感染可以被靶向治疗，像单克隆抗体不需要有实质性的血脑屏障穿透。本申请的修订目的如下：目的1：鉴定出具有较强抗人PrPSc复制治疗作用的单克隆抗体，并将其人源化，用于临床试验和应用。抗prp单克隆抗体具有独特的治疗效果。我们在小鼠朊病毒感染的细胞培养模型中鉴定出三种单克隆抗体：6D11、7H6和7A12， IC50%<1¿g/ml，能够永久消除PrPSc。在我们的初步研究中，我们已经证实了这些单克隆抗体与人类PrP的反应性，因此我们计划使用我们正在开发的人类朊病毒感染性细胞培养模型来表征它们的治疗效果。我们的测试还将包括Mab 3F4，它与人类PrPSc强烈反应，其抗原表位位于PrP序列的中心部分，这是其他治疗性Mab的一个特点。目的二将解决被动免疫是否可以预防朊病毒感染的神经系统后果的基本问题。我们有初步的数据表明，用Mab-6D11免疫8周，可以抑制PrPSc在外中枢神经系统感染的野生型小鼠淋巴器官中的复制，并将潜伏期延长约40%，改善脑病理。目标II中概述的大多数实验将在小鼠朊病毒感染模型中进行，使用在细胞培养模型中对小鼠PrPSc有效的单克隆抗体。如果在目的1中成功鉴定出具有治疗人PrPSc复制作用的单克隆抗体，则将在鼠PrP敲除背景下表达人PrP129M的转基因小鼠中进行测试，这些小鼠易受人朊病毒感染。修订目的III的目的是提供单克隆抗体治疗效果的机制见解。Aim III概述了一组实验，研究在获得痒病形式特性的哪个阶段，治疗性抗prp单克隆抗体会干扰PrPSc的形成，以及它们是否也促进PrPSc的降解。此外，我们将确定单克隆抗体活性发生的细胞区室。这项拨款提案的总体目标是为开发人类朊病毒感染的被动免疫疗法提供基础。公共卫生相关性：该项目的目标是开发一种被动免疫方法，以防止意外暴露于朊病毒的人类发展为普遍致命的疾病。朊病毒是一种传染性神经退行性疾病，目前尚无有效的治疗方法。此外，朊病毒被认为是潜在的生物恐怖主义制剂。

DESCRIPTION (provided by applicant): The main research hypothesis in this grant proposal is that passive immunization with anti-prion monoclonal antibodies (Mabs) can effectively prevent neurological consequence resulting from extra- CNS prion infection. Prion diseases (prionoses) are transmissible, invariably fatal, neurodegenerative diseases associated with a conformational transformation of cellular prion protein PrPC into a toxic, infectious, and self-replicating PrPSC conformer for which no effective treatment is currently available. Accidental prion infection in humans may occur as a result of blood transfusion or organ transplant, ingestion of prion contaminated food, or use of prion contaminated surgical instruments. Following extra-CNS exposure, PrPSc replicates within the lymphoid organs for months to years before CNS invasion causes clinical symptoms. Therefore, prion infection in the pre-CNS stage can be targeted by therapeutics, which like Mabs are not required to have substantial blood-brain-barrier penetration. Revised aims of this grant proposal are as follows: Aim I will identify Mabs with strong therapeutic effect against human PrPSc replication, which can be humanized for clinical testing and application. Anti-PrP Mabs with strong therapeutic effect are unique. We have identified three Mabs: 6D11, 7H6, and 7A12 capable of permanent PrPSc abrogation in a cell culture model of murine prion infectivity with IC50%<1¿g/ml. In our preliminary studies, we have confirmed reactivity of these Mabs with human PrP and thereby we are planning to characterize their therapeutic effect using cell culture model of human prion infectivity which we are developing. Our testing will also include Mab 3F4, which strongly reacts with human PrPSc and has its antigen epitope located in the central portion of PrP sequence, a characteristic of other therapeutic Mabs. Aim II will address the fundamental question of whether passive immunization can prevent neurological consequence of prion infection. We have preliminary data to demonstrate that eight-week immunization with Mab-6D11 suppresses PrPSc replication in the lymphoid organs of extra-CNS infected wild type mice and the treatment extends the incubation period by ~40% and ameliorates brain pathology. The majority of experiments outlined in aim II will be carried out in murine models of prion infections using Mabs effective against murine PrPSc in cell culture models. If Mabs with therapeutic effect against human PrPSc replication are successfully identified in aim I, they shall be tested in transgenic mice expressing human PrP129M on murine PrP knock-out background which are susceptible to infection with human prions. The goal of revised aim III is to provide a mechanistic insight into therapeutic effect of Mabs. Aim III outlines a set of experiments investigating at what stage(s) of acquisition of scrapie form properties therapeutic anti-PrP Mabs interfere with PrPSc formation and whether they also facilitate PrPSc degradation. Furthermore, we will determine the cellular compartment(s) wherein Mabs activity occurs. The overall goal of this grant proposal is to provide a foundation for development of a passive immunization therapy for human prion infection. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a passive immunization approach to prevent humans accidentally exposed to prions from developing a universally fatal disease. Prionoses are transmissible neurodegenerative diseases for which there is no effective therapy. Moreover, prions are considered a potential bioterrorism agent.