Therapeutic Monoclonal Antibodies for Human Prion Diseases

描述(申请人提供)：这项拨款提案中的主要研究假设是，用抗Prion单抗(MAbs)被动免疫可以有效地预防中枢神经系统外Prion感染所导致的神经后果。Prion病(Prionoses)是一种遗传性、致命性、神经退行性疾病，与细胞PrPC蛋白构象转化为一种有毒、有感染性和自我复制的PrPSC构象有关，目前还没有有效的治疗方法。人类可能因输血或器官移植、摄入被病毒污染的食物或使用被病毒污染的手术器械而发生意外的病毒感染。在中枢神经系统外暴露后，PrPSc在淋巴器官内复制数月至数年，然后中枢神经系统入侵引起临床症状。因此，中枢神经系统前阶段的Prion感染可以成为治疗的靶点，与单抗一样，治疗不需要有实质性的血脑屏障穿透。这项拨款提案的修订目标如下：目的I将筛选出对人类PrPSc复制有较强治疗作用的单抗，使其人源化，用于临床测试和应用。抗PrP单抗是独一无二的，具有很强的治疗效果。在IC50%和1g/ml的小鼠PrP感染性细胞培养模型中，我们鉴定了三株能够永久清除PrPSc的单抗：6D11、7H6和7A12。在我们的初步研究中，我们证实了这些单抗与人PrP的反应性，因此我们计划用我们正在开发的人PrP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单抗3F4，它与人PrPSc发生强烈反应，其抗原表位位于PrP序列的中央部分，这是其他治疗性单抗的特征。目的II将解决被动免疫是否可以防止Pron感染的神经系统后果这一根本问题。我们有初步的数据表明，用Mab-6D11免疫8周后，抑制了CNS外感染野生型小鼠淋巴器官中PrPSc的复制，并延长了潜伏期约40%，并改善了脑病理。在AIM II中概述的大多数实验将在普恩病毒感染的小鼠模型中进行，在细胞培养模型中使用有效对抗小鼠PrPSc的单抗。如果在AIM I中成功鉴定出对人类PrPSc复制具有治疗作用的单抗，则应在表达人PrP129M的转基因小鼠身上进行测试，该转基因小鼠在小鼠PrP基因敲除背景下表达人PrP129M，而小鼠对人PrP感染易感。修订的目标III的目标是提供对单抗治疗效果的机械性洞察。目的III概述了一组实验，调查在获取瘙痒病的哪个阶段(S)，治疗性抗PrP单抗干扰PrPSc的形成，以及它们是否也促进PrPSc的降解。此外，我们将确定发生单抗活性的细胞室(S)。这项赠款提案的总体目标是为开发一种针对人类普恩病毒感染的被动免疫疗法提供基础。与公共卫生的相关性：该项目的目标是开发一种被动免疫方法，以防止意外接触普恩病毒的人类患上一种普遍致命的疾病。脊髓炎是一种传染性神经退行性疾病，目前还没有有效的治疗方法。此外，Prion被认为是潜在的生物恐怖主义毒剂。

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.