Molecular Analysis of Human Anti-GBM Antibodies

描述(申请人提供)：抗肾小球基底膜抗体(GBM)介导的肾小球肾炎是由针对肾小球固有成分的致病抗体所产生的人类肾小球疾病的原型。尽管人们对该病的靶标基底膜抗原(Ag)和炎性介质的鉴定进行了大量的研究，但对其结构特征、免疫生物学和编码致病性人抗GBM自身抗体的基因的研究较少。然而，最近的技术进步使人自身抗体的筛选和大规模生产成为可能。这些进展，再加上人抗GBM抗体的主要靶标自体抗原(α3型IV型胶原，α3(IV))的鉴定、克隆、表达和可获得性，为确定人抗GBM抗体的遗传和结构特征提供了理想的环境、必要的方法和分子试剂。在过去的项目期间，我们利用这些方法和其他方法检测了人抗GBM抗体，结果表明来自不同患者的自身抗体具有高亲和力和共同的结构特征。此外，在Xenomouse II中开发了一种新的抗GBM疾病模型，Xenomouse II是一种表达人免疫球蛋白而不是小鼠免疫球蛋白的新型小鼠品系。在(α3(IV))免疫后，小鼠出现由人自身抗体介导的新月体肾小球肾炎，其结构特征与患者产生的自身抗体相同。此外，还鉴定并鉴定了一株致病性的单抗。同时，来自肾病患者的FAB的遗传和结构分析已经开始。我们的目标是将这些研究扩展到检测来自患者和异种鼠II的自身抗体，以追求以下具体目标：i)确定致病的人抗α3(IV)胶原抗体的结构特性，并利用这些信息开发具有高亲和力和特异性的人抗GBM抗体的小分子。我们的工作假设是，对致病自体抗体的分子和结构分析，结合与α3(IV)相关致病表位的现有信息，将导致对这种特殊的自体抗体-自体抗原相互作用的更准确的理解，并且这些信息，再加上已建立的人抗GBM抗体介导的肾炎模型，将有助于设计和评估自体抗体沉积或/和自体抗体产生的特定抑制剂。这一结果也有可能为深入了解致病性人类自身抗体的起源提供线索。此外，由于抗GBM疾病患者的自体抗体反应集中在特定的并将定义的自体抗原(Alpha3(IV))，这种情况代表了一种理想的情况，可以评估针对免疫反应细胞上的可溶性和表面受体的特定子集的治疗，以改变疾病的活动性。如果成功，该策略有可能应用于开发其他抗体介导的疾病的治疗策略。

DESCRIPTION (provided by applicant): Glomerulonephritis mediated by antibodies (Ab) to the glomerular basement membrane (GBM) is the prototype of human glomerular disease produced by pathogenic Ab to intrinsic glomerular components. Although there has been intense investigation into identification of the target basement membrane antigen (Ag) and inflammatory mediators of this disease, less attention has been devoted to the structural features, immunobiology and genes encoding pathogenic human anti-GBM autoAb. Recent technical advances, however, make the selection and large scale production of human autoAb feasible. These advances, coupled with the identification, cloning, expression and availability of the major target autoAg (alpha3 type IV collagen, (alpha3 (IV) for human anti-GBM Ab, provide the ideal circumstances, necessary methodologies and molecular reagents to define the genetic and structural features of human anti-GBM Ab. In the past project period, we have utilized these and other approaches to examine human antiGBM Ab, and the results have defined autoAb, from different patients, as having high affinity and with shared structural features. Furthermore, a new model of anti-GBM disease was developed in Xenomouse II, a novel murine strain that expresses human, but not murine, immunoglobulins. Following immunization with (alpha3(IV), the mice develop crescentic glomerulonephritis mediated by human autoAb that share structural features with autoAb produced by patients. Further-more a pathogenic monoclonal Ab have been identified and characterized. In parallel genetic and structural analysis of Fab derived from nephritic patients has been initiated. Our goal is to extend these studies to examine autoAb from both patients and Xenomouse II to pursue the following Specific Aims: i) To define the structural properties of pathogenic human anti-alpha3 (IV) collagen Ab.and use this information ii) To develop small molecules with high affinity and specificity for human anti-GBM Ab. Our working hypothesis is that molecular and structural analysis of pathogenic autoAb, together with existing information pertaining to the relevant pathogenic epitopes of alpha3(IV) will lead to a more precise understanding of this particular autoAb-autoAg interaction, and that this information, coupled with an established model of nephritis mediated by human anti-GBM Ab, will be useful in the design and evaluation of specific inhibitors of autoAb deposition or/and autoAb production. The results also have the potential to provide insight into the origins of pathogenic human autoAb. Furthermore, since the autoAb response in patients with anti-GBM disease is focused on a specific and will defined autoAg (alpha3(IV), this condition represent the ideal situation, where evaluation of therapy directed against a specific subset of soluble and surface receptors on immunoreactive cells, to alter disease activity, can be evaluated. If successful, the strategy has the potential for application in the development of therapeutic strategies in other Ab-mediated diseases.