Molecular Analysis of Human Anti-GBM Antibodies

描述（由申请人提供）：肾小球基底膜（GBM）抗体（Ab）介导的肾小球肾炎是由致病性Ab对肾小球固有成分产生的人类肾小球疾病的原型。虽然人们对该病的靶基底膜抗原（Ag）和炎症介质的鉴定进行了大量研究，但对致病性人抗gbm自身抗体的结构特征、免疫生物学和编码基因的关注较少。然而，最近的技术进步使得人类自体抗体的选择和大规模生产成为可能。这些进展，再加上人类抗gbm抗体的主要靶点autoAg （alpha3型IV胶原，alpha3 (IV)）的鉴定、克隆、表达和可用性，为确定人类抗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来检测人类抗gbm抗体，结果定义了来自不同患者的autoAb。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是一种表达人类而非小鼠免疫球蛋白的新型小鼠菌株。用(alpha3（IV）免疫后，小鼠发生月牙状肾小球肾炎，由人自身抗体介导，与患者自身抗体具有相同的结构特征。此外，还鉴定和鉴定了一种致病性单克隆抗体。同时，从肾病患者中提取的Fab基因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Xenomouse II的autoAb，以实现以下具体目标：ii)开发具有高亲和力和特异性的人抗gbm抗体小分子。我们的工作假设是，对致病性autoAb的分子和结构分析，以及与alpha3（IV）相关的致病表位相关的现有信息，将导致对这种特殊的autoAb- autoag相互作用的更精确的理解，并且这些信息，结合人类抗gbm抗体介导的肾炎模型，将有助于设计和评估自身抗体沉积或/和自身抗体产生的特异性抑制剂。该结果也有可能为了解致病性人类自身抗体的起源提供线索。此外，由于抗gbm疾病患者的自身抗体反应集中在一种特定的、将被定义的自身抗原（alpha3(IV)）上，这种情况代表了一种理想的情况，在这种情况下，可以评估针对免疫反应细胞上的一种特定的可溶性和表面受体亚群的治疗，以改变疾病的活动性。如果成功，该策略有潜力应用于其他抗体介导疾病的治疗策略的开发。

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.