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

• 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
• 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

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。这个 最常见的亚型特征是针对乙酰胆碱受体(AChR)的自身抗体(AutoAbbs)， 在神经肌肉连接处的肌肉终板上表达一种膜通道。病理是直接的 由于AChR AutoAbs。患者血清中的AChR自体抗体具有广泛的异质性 特异性，因为它包括识别AChR的四个不同亚基(a、b、e或d)中的每一个的自动抗体。 此外，AChR AutoAbbs可以使用三种不同的机制来影响病理：(I)补体导向 组织损伤，(Ii)阻断乙酰胆碱的结合部位和(Iii)调节(内化)AChR。 虽然AChR自体抗体是致病的，但不同患者之间和个体内的滴度差别很大。 重要的是，滴度与临床严重程度没有很好的相关性，也不能预测 治疗结果。此外，针对AChR AutoAb的新的生物治疗策略 补体激活或降低循环滴度在反应中表现出很大的变异性，包括 不要回应。滴度和疾病严重程度之间的分离，以及对 自身抗体靶向治疗还不是很清楚。我们知识中的这种鸿沟首先存在是因为AChR 自体抗体通常用患者血清进行研究，而患者血清被复杂的特异性混合所混淆。 第二，现有的检测方法只检测AChR自身抗体的结合，而不是它们的致病能力或机制。 我们将通过以下方式解决我们知识中的这些空白：(I)产生罕见的人类单克隆AChR AutoAbb，(Ii) 使用新的方法来测量自身抗体致病的不同机制，以及(Iii)将 具有致病能力的自身抗体特性。为此，我们采用了无偏见的高吞吐量方法 通过克隆B细胞生产AChR特异性人单抗，从而绕过了相关的技术挑战 通过克隆以该多亚单位膜通道为靶点的单抗。这将负担得起生产AChR特定的 单抗文库，它包括识别不同亚基和这些亚基中的表位的单抗。新的 研究AChR自身抗体致病的不同效应机制的方法也已开发出来。 我们将检测AChR自体抗体介导的乙酰胆碱阻断和AChR的调节功能 单抗。重要的是，一种用于测量补体激活的试验，其中补体抑制物(CD46， CD55和CD59)被敲除，将用于检测AChR自身抗体依赖的补体活性。这些 实验将确定AChR自动抗体的特异性和分子特性如何与 致病效应器功能。总体而言，这项调查将(I)提供一套具有良好特征的mAbs，它将 作为更准确地模拟AChR AutoAb病理的工具；(Ii)提供一个理解框架 表位特异性和补体激活之间的关系以及(Iii)寻找潜在的新治疗方法 通过特异性靶向AChR自身抗体致病机制治疗MG的途径