Pathogenic Autoantibodies with Specificity for Aberrant Glycoproteins: Assessment of a Therapeutic Target in an Autoimmune Disease

伊加肾病（IgAN）是世界上最常见的肾小球肾炎，其导致终末期肾病， 20-40%的患者，并可减少预期寿命长达10年，因为没有已知的治疗或疾病- 具体治疗。大多数IgAN患者，无论年龄和种族，都有免疫缺陷，导致 产生致病性的含IgA 1的免疫复合物，其最终存款在肾脏中以诱导 肾损伤这些肾脏免疫沉积物可能来源于由IgA 1组成的循环免疫复合物 与Gd-IgA 1特异性IgG自身抗体结合的铰链区半乳糖缺陷O-聚糖（Gd-IgA 1）。的 该项目的长期目标是确定导致致病性疾病形成的潜在机制。 免疫复合物，以便可以开发IgAN特异性治疗。我们的假设是一种分子- IgAN患者Gd-IgA 1特异性IgG自身抗体的水平表征与原子水平 免疫复合物中自身抗体的表征将大大促进我们对 IgAN中免疫复合物形成。这些信息将反过来为开发新的 疾病特异性治疗。在过去的三年里，调查人员的实验室利用了 生物化学、分子、结构和临床研究，以开始IgAN特异性 自身抗体我们已经证明，来自IgAN患者的IgG自身抗体具有一个序列（氨基）， - 在重链可变部分（VH）中框架3和CDR 3的连接处的氨基酸YCSR/K）， 其中丝氨酸残基对于Gd-IgA 1结合是必需的。这种丝氨酸残基来源于体细胞 在一些实施方案中，Vh基因突变是由高突变（Ala->Ser）引起的，而不是来自Vh种系基因的遗传突变。我们的晶体学研究 用IgAN衍生的（YCSK）和生殖系回复的（YCAK）重组IgG自身抗体显示， 氨基酸序列上的这种看似微小的差异对蛋白质周围的元素具有变构效应。 丝氨酸残基，产生与CDR环并置的新表面。这个表面是一个潜在的结合位点 Gd-IgA 1铰链区糖肽的一部分，是设计IgG自身抗体的潜在靶点 抑制剂的在本提案中，我们将确定IgG的群体和个体水平的变异性， 基于VH/VL序列和Gd-IgA 1结合的IgAN中的自身抗体（Aim 1），确定了结构 代表性IgG自身抗体的特征和Gd-IgA 1识别的分子机制（Aim 2）， 并开发阻断IgG自身抗体与Gd-IgA 1结合的方法（目的3）。通过利用我们 获得生物库临床样本、新患者、新的高通量克隆方法， 特异性Gd-IgA 1的IgG自身抗体的表达，用于结构分析的高分辨率方法，以及高分辨率的免疫球蛋白的表达。 通过抑制剂的通量测试，我们的研究已经进展到一个阶段， 这些自身抗体将促进我们对IgAN疾病驱动机制的理解。的结果 我们的研究将确定疾病特异性靶点以防止IgAN中致病性免疫复合物的形成。

IgA nephropathy (IgAN), the most common glomerulonephritis worldwide, leads to end-stage renal disease in 20-40% of patients and can reduce life expectancy by up to 10 years, as there is no known cure or disease- specific treatment. Most IgAN patients, regardless of age and ethnicity, have immunologic defects resulting in generation of pathogenic IgA1-containing immune complexes, which ultimately deposit in the kidneys to induce renal injury. These renal immunodeposits likely originate from circulating immune complexes consisting of IgA1 with hinge-region galactose-deficient O-glycans (Gd-IgA1) bound by Gd-IgA1-specific IgG autoantibodies. The long-term goal of this project is to define the underlying mechanisms that lead to the formation of pathogenic immune complexes, so that IgAN-specific treatments can be developed. Our hypothesis is that a molecular- level characterization of Gd-IgA1-specific IgG autoantibodies from IgAN patients coupled with an atomic-level characterization of autoantibodies in immune complexes will significantly advance our understanding of immune-complex formation in IgAN. This information will in-turn provide a basis for development of new disease-specific treatments. Over the past three years, the laboratories of the investigators have utilized biochemical, molecular, structural, and clinical studies to begin characterization of IgAN-specific autoantibodies. We have shown that IgG autoantibodies from patients with IgAN harbor a sequence (amino acids YCSR/K) at the junction of framework 3 and the CDR3 in the variable part of the heavy chain (VH), wherein the serine residue is essential for Gd-IgA1 binding. This serine residue originates from a somatic hypermutation (Ala->Ser) and not from a genetic mutation of a VH germline gene. Our crystallographic studies with an IgAN-derived (YCSK) and a germline-reverted (YCAK) recombinant IgG autoantibody revealed that this seemingly minor difference in the amino-acid sequence had allosteric effects on elements surrounding the serine residue, generating a new surface juxtaposed to the CDR loops. This surface is a potential binding site for part of the Gd-IgA1 hinge-region glycopeptide and is a potential target for the design of IgG autoantibody inhibitors. In this proposal, we will determine the population- and individual-level variability of IgG autoantibodies in IgAN based on VH/VL sequences and Gd-IgA1 binding (Aim 1), determine the structural features of representative IgG autoantibodies and the molecular mechanism of Gd-IgA1 recognition (Aim 2), and develop approaches to block the binding of IgG autoantibodies to Gd-IgA1 (Aim 3). By leveraging our access to biobanked clinical samples, new patients, the new high-throughput approaches for cloning and expression of IgG autoantibodies specific Gd-IgA1, high-resolution methods for structural analyses, and high- throughput testing of inhibitors, our studies have progressed to a stage where molecular-level assessments of the autoantibodies will advance our understanding of the mechanisms that drive IgAN disease. The results of our studies will define disease-specific targets to prevent pathogenic immune-complex formation in IgAN.