Molecular Analysis of Human Anti-GBM Antibodies

描述（申请人提供）：抗肾小球基底膜（GBM）抗体（Ab）介导的肾小球肾炎是由抗内源性肾小球成分的致病性Ab产生的人类肾小球疾病的原型。虽然已经有大量的研究，以确定目标基底膜抗原（Ag）和炎症介质的这种疾病，较少关注的结构特征，免疫生物学和编码致病性人抗GBM自身抗体的基因。然而，最近的技术进步使得人类自身抗体的选择和大规模生产成为可能。这些进展，再加上人抗GBM Ab的主要靶点autoAg（α 3 IV型胶原，α 3（IV））的鉴定、克隆、表达和可用性，为确定人抗GBM Ab的遗传和结构特征提供了理想的环境、必要的方法学和分子试剂。在过去的项目期间，我们已经利用这些和其他方法来检查人抗GBM抗体，结果已经定义了autoAb，从不同的患者，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是一种表达人而非鼠免疫球蛋白的新型鼠品系。在用α 3（IV）免疫后，小鼠发生由人自身抗体介导的新月体肾小球肾炎，所述人自身抗体与患者产生的自身抗体具有相同的结构特征。此外，还鉴定和表征了一种致病性单克隆抗体。与此同时，已开始对来自肾病患者的Fab进行遗传和结构分析。我们的目标是将这些研究扩展到检查来自患者和Xenomouse II的autoAb，以实现以下特定目的：i）确定致病性人抗α 3（IV）胶原抗体的结构特性，并使用此信息ii）开发对人抗GBM抗体具有高亲和力和特异性的小分子。我们的工作假设是，致病性自身抗体的分子和结构分析，以及与α 3（IV）的相关致病性表位有关的现有信息，将导致对这种特定的自身抗体-自身抗原相互作用的更精确的理解，并且该信息，加上由人抗GBM抗体介导的肾炎的已建立模型，将可用于设计和评价自身抗体沉积或/和自身抗体产生的特异性抑制剂。这些结果也有可能为了解致病性人类自身抗体的起源提供帮助。此外，由于抗GBM疾病患者的自身抗体应答集中于特定的和将定义的自身抗原（α 3（IV）），因此这种情况代表了理想的情况，其中可以评价针对免疫反应性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活性。如果成功的话，该策略具有在其他Ab介导的疾病的治疗策略的开发中应用的潜力。

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.