IG REPERTOIRE OF COMBINATORIAL AUTOANTIBODIES IN PBC

PBC 中组合自身抗体的 IG 库

• 批准号: 2066563
• 负责人: PATRICK S LEUNG
• 金额: $ 9.69万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2066563
• 关键词: aldehyde /ketone oxidoreductase; autoantibody; autoantigens; autoimmune disorder; epitope mapping; gene rearrangement; genetic library; human tissue; immunoglobulin genes; molecular cloning; monoclonal antibody; pathologic process; primary biliary cirrhosis; protein sequence; pyruvate dehydrogenase

The generation and characterization of human monoclonal antibodies, in contrast to murine systems, has been difficult and with few exceptions, non-productive. In fact, current hypotheses of Ig gene usage in human organ specific autoimmune diseases are either extrapolated from murine lupus or based on analysis of rheumatoid factors. My laboratory has been successful in generating combinatorial autoantibodies derived from the lymph node of patients with primary biliary cirrhosis (PBC). These Fab autoantibodies are directed at PDC-E2, the major autoantigen of PBC. Moreover, the nucleotide sequence of our combinatorial autoantibodies suggest that these antibodies are clonally related and reflect a restricted response to PDC-E2. Moreover, the V-H genes show considerable somatic mutation in CDR and three of these antibodies utilize a V-H germline gene that has not hitherto been published. PBC is one of the few autoimmune diseases for which a) the autoantigen and dominant epitope have been identified; b) the genes coding for these autoantigens have been cloned and sequenced; and c) recombinant proteins and synthetic peptides are available. In this First Award I will take advantage of these strengths and address several key questions. For example, we will obtain additional data on the Ig gene usage encoding autoantibodies to PDC-E2 and BCKD-E2: more than 90% of patients with PBC react with one or more of these autoantigens. We will also determine, as our pilot data suggests, that these autoantibodies are encoded by a restricted response, reflect uncommon gene usage, and have undergone somatic mutation. Additionally, we will determine similarities which exist between the coding sequences of autoantibodies to PDC-E2 compared to BCKD-E2. Finally, we will take advantage of our random peptide library (peptide on plasmid) and determine the autoepitopes recognized by these autoantibodies. We will compare such data to epitopes determined by overlapping recombinant peptides. Such data is important because by confocal microscopy we have demonstrated that our combinatorial autoantibodies stain a unique protein located only on the luminal side of bile duct epithelial cells in patients with PBC but not PSC. Further data on epitope recognition and mapping may shed further light on this issue. The answers to these issues will have significant implications not only for PBC, but also for other organ specific autoimmune diseases and will provide significant data on the utility of combinatorial autoantibodies.

人类单克隆抗体的产生和表征 与鼠系统形成鲜明对比，很困难，很少 例外，非生产性。 实际上，Ig基因的当前假设 人体器官特异性自身免疫性疾病的使用是 外推从鼠狼疮或基于类风湿的分析 因素。 我的实验室已经成功产生 源自患者淋巴结的组合自身抗体 与原发性胆道肝硬化（PBC）。 这些Fab自身抗体是 针对PDC-E2，PDC-E2是PBC的主要自动抗原。 而且， 我们组合自身抗体的核苷酸序列表明 这些抗体是克隆相关的，反映了受限制的 对PDC-E2的响应。 此外，V-H基因显示出相当大的体细胞 CDR中的突变，其中三种抗体利用V-H系 迄今尚未出版的基因。 PBC是少数几个 a）自身免疫性疾病a）自身抗原和显性表位 已经确定； b）编码这些自动抗原的基因具有 被克隆和测序； c）重组蛋白和合成 肽可用。 在第一个奖项中，我将利用 这些优势并解决了几个关键问题。 例如，我们 将获得有关IG基因使用的其他数据编码 PDC-E2和BCKD-E2的自身抗体：超过90％的患者 PBC与其中一种或多种自身抗原反应。我们也会 正如我们的飞行员数据所表明的那样，确定这些自身抗体是 由限制响应编码，反映了罕见的基因使用情况，并 经历了躯体突变。 此外，我们将确定 在编码序列之间存在的相似性 与BCKD-E2相比，PDC-E2的自身抗体。 最后，我们会接受 我们随机肽文库（质粒上的肽）和 确定这些自动抗体识别的自动发言。 我们 将将此类数据与通过重叠确定的表位进行比较 重组肽。 这样的数据很重要，因为通过共享 显微镜我们已经证明了我们的组合自身抗体 染色仅位于胆管腔侧的独特蛋白质 PBC患者但不是PSC的上皮细胞。 进一步的数据 表位识别和映射可能会进一步阐明这个问题。 这些问题的答案将具有重要的影响 仅适用于PBC，也适用于其他特定器官的自身免疫性疾病 并将提供有关组合效用的重要数据 自动抗体。