Molecular determinants driving diverse mechanisms of antibody-mediated pathology

驱动抗体介导病理学多种机制的分子决定因素

基本信息

批准号：
10312209
负责人：
Kevin C O'connor
金额：
$ 25.13万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-20 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10312209
关键词：
Acetylcholine Address Adopted Affect Antibodies Antigens Autoantibodies Autoimmune Autoimmune Diseases Automobile Driving B-Lymphocytes Binding Binding Sites Biological Assay Biological Response Modifier Therapy CD46 Antigen CRISPR/Cas technology Cell Line Cholinergic Receptors Classical Complement Pathway Clinical Cloning Collection Communication Complement Complement Activation Complement Inactivators Complex Complex Mixtures Consequentialism Detection Diagnosis Disease Disease Progression Dissection Epitopes Frequencies Genes Heterogeneity Human Impairment Individual Investigation Ion Channel Knock-out Knowledge Laboratories Libraries Link Mammalian Cell Measures Mediating Modeling Molecular Monitor Monoclonal Antibodies Muscle Muscle Weakness Myasthenia Gravis Nerve Neuromuscular Diseases Neuromuscular Junction Neuromuscular Junction Diseases Neuromyelitis Optica Nicotinic Receptors Pathogenicity Pathology Patients Prediction of Response to Therapy Prevalence Production Property Public Health Recombinants Resources Series Serum Severities Severity of illness Somatic Mutation Specificity Technology Testing Tissues Treatment outcome crosslink experience experimental study human monoclonal antibodies neuromuscular neuromuscular transmission novel novel strategies novel therapeutics pathogenic autoantibodies personalized therapeutic prevent receptor response sample fixation tool treatment response variable region gene

项目摘要

Project Summary. Myasthenia gravis (MG) is an autoimmune disorder of neuromuscular transmission. The most common subtype is characterized by autoantibodies (autoAbs) targeting the acetylcholine receptor (AChR), a membrane channel expressed on the muscle end-plate at the neuromuscular junction. The pathology is directly due to the AChR autoAbs. The AChR autoAbs in the serum of patients are broadly heterogeneous in their specificity as it includes autoAbs that recognize each of the four different subunits (a, b, e or d) of AChR. Furthermore, AChR autoAbs can use three distinct mechanisms to effect pathology: (i) complement-directed tissue damage, (ii) blocking the binding site for acetylcholine and (iii) modulation (internalization) of the AChR. Although AChR autoAbs are pathogenic, titers vary widely between different patients and within individuals during the course of their disease; importantly, titer neither correlates well with clinical severity nor predicts treatment outcome. Furthermore, new biological therapeutic strategies that specifically target AChR autoAb complement activation or reduce circulating titers have shown wide variability in response including patients that do not respond. The disassociation between titer and disease severity, and the heterogeneous response to autoantibody targeting treatments are not well understood. This gap in our knowledge exists first because AChR autoAbs are often studied with patient serum, which are confounded by the complex mixture of specificities. Second, existing assays only measure AChR autoAb binding, but not their pathogenic capacity or mechanism. We will address these gaps in our knowledge by (i) generating rare human monoclonal AChR autoAbs, (ii) using novel approaches to measure the different mechanisms of autoAb pathogenicity, and (iii) associating the autoAb properties with pathogenic capacity. To this end, we applied an unbiased high-throughput approach for producing AChR-specific human mAbs by cloning B cells, which circumvented technical challenges associated with cloning mAbs targeting this multi-subunit membrane channel. This will afford production of an AChR specific mAb library, which includes mAbs that recognize different subunits and epitopes within those subunits. New approaches to study the different effector mechanisms of AChR autoAb pathogenicity have also been developed. We will measure AChR autoAb-mediated acetylcholine blocking and AChR modulating functions of the AChR mAbs. Importantly, an assay for measuring complement activation, in which the complement inhibitors (CD46, CD55 and CD59) are knocked out, will be used to test AChR autoAb-dependent complement activity. These experiments will define how the specificity and molecular properties of AChR autoAb are associated with pathogenic effector function. Overall, this investigation will (i) provide a set of well-characterized mAbs which will serve as tools for more accurate modeling of AChR autoAb pathology; (ii) provide a framework for understanding the association between epitope specificity and complement activation and (iii) identify potential new therapeutic avenues for treating MG through specifically targeting AChR autoAb pathogenic mechanisms.

项目摘要。重症肌无力（MG）是一种神经肌肉传递的自身免疫性疾病。这最常见的亚型的特点是针对乙酰胆碱受体 (AChR) 的自身抗体 (autoAbs)，在神经肌肉接头处的肌肉终板上表达的膜通道。病理直接是由于 AChR 自动抗体。患者血清中的 AChR 自体抗体在其性质上存在广泛的异质性特异性，因为它包括识别 AChR 四个不同亚基（a、b、e 或 d）中每一个的 autoAb。此外，AChR autoAbs 可以使用三种不同的机制来影响病理学：（i）补体定向组织损伤，(ii) 阻断乙酰胆碱的结合位点，以及 (iii) AChR 的调节（内化）。尽管 AChR 自身抗体具有致病性，但不同患者和个体内部的滴度差异很大在患病期间；重要的是，滴度既不能很好地与临床严重程度相关，也不能预测治疗结果。此外，专门针对 AChR autoAb 的新生物治疗策略补体激活或降低循环滴度已显示出反应的广泛差异，包括以下患者：不回应。滴度和疾病严重程度之间的分离，以及对疾病的异质反应自身抗体靶向治疗尚不清楚。我们的知识差距首先存在是因为 AChR 自身抗体经常用患者血清进行研究，这会因复杂的特异性混合而变得混乱。其次，现有的检测方法仅测量 AChR autoAb 结合，而不测量其致病能力或机制。我们将通过 (i) 生成稀有的人类单克隆 AChR autoAbs，(ii) 使用新方法来测量自身抗体致病性的不同机制，以及（iii）将具有致病能力的自身抗体特性。为此，我们采用了无偏高通量方法通过克隆 B 细胞生产 AChR 特异性人类单克隆抗体，从而规避了相关的技术挑战以及针对该多亚基膜通道的克隆单克隆抗体。这将提供 AChR 特异性的生产 mAb 库，其中包括识别不同亚基和这些亚基内表位的 mAb。新的还开发了研究 AChR autoAb 致病性不同效应机制的方法。我们将测量 AChR autoAb 介导的乙酰胆碱阻断和 AChR 调节功能单克隆抗体。重要的是，一种测量补体激活的测定，其中补体抑制剂（CD46， CD55 和 CD59) 被敲除，将用于测试 AChR autoAb 依赖性补体活性。这些实验将确定 AChR autoAb 的特异性和分子特性如何与致病效应功能。总体而言，这项研究将 (i) 提供一组特征良好的单克隆抗体，这些单克隆抗体将作为 AChR autoAb 病理学更准确建模的工具； (ii) 提供一个理解框架表位特异性和补体激活之间的关联，以及（iii）确定潜在的新治疗方法通过专门针对 AChR autoAb 致病机制来治疗 MG 的途径。