Cloning and genetics of human pemphigus autoantibodies

描述(申请人提供)：寻常型天疱疮和叶疱疮是一种严重的、有时危及生命的自身免疫性水疱性皮肤病，在这些皮肤病中，抗桥粒蛋白(DSG)3和1的自身抗体、桥粒黏附分子会导致角质形成细胞黏附的丧失。最终，更具体的治疗和诊断方法将需要对致病自身抗体进行更详细的表征。通过确定天疱疮患者个体的遗传特性和良好的特异性，即来自天疱疮患者的单抗(MAbs)，可以解决关于常见免疫球蛋白可变片段基因的使用以及患者之间共享的致病独特型和表位的问题。一种名为噬菌体展示的强大分子技术将被用于克隆和鉴定人类抗DSG自身抗体。这项技术将允许检验以下假设：a)来自天疱疮患者的抗DSG1和Dsg3的单抗既是致病的，也是非致病的，并且这两种类型都可以被分离出来；b)抗DSG抗体中存在受遗传限制的重链和轻链使用；c)致病单抗在Dsg3和DSG1的氨基末端结合一组受限的表位；d)不同的患者共享致病自身抗体独特型；以及e)致病独特型可以被模拟Dsg3或DSG1致病表位的肽所阻断。抗体将通过在Dsg3和DSG1上的选择从患者的噬菌体展示文库中克隆出来。用酶联免疫吸附试验、免疫荧光法和Western blotting对所制备的人源单抗进行鉴定。将对单抗进行致病性测试，然后比较致病抗体和非致病抗体的遗传学。用致病单抗筛选噬菌体多肽文库。将绘制DSG上各自的表位图，并定义(患者内部和患者之间的)交叉反应独特型。这些实验的结果将大大增加我们对天疱疮中人类自身抗体的了解。该项目将：确定不同的人类抗体如何在这种疾病的病理中起作用，为研究这些和其他皮肤病的其他研究人员开发有价值的人类单抗试剂，并将指出解决这些潜在威胁生命的疾病的致病抗体的靶向治疗和诊断的新方法。

DESCRIPTION (provided by applicant): Pemphigus vulgaris and foliaceus are severe, sometimes life-threatening, autoimmune blistering skin diseases in which autoantibodies against desmoglein (Dsg) 3 and 1, desmosomal adhesion molecules, cause loss of keratinocyte cell adhesion. Ultimately, more specific treatments and diagnostic methods will require more detailed characterization of disease-causing autoantibodies. By determining the genetic properties and fine specificities of individual, i.e. monoclonal, antibodies (mAbs) from pemphigus patients, questions regarding common immunoglobulin variable segment gene usage, and shared pathogenic idiotypes and epitopes among patients can be addressed. A powerful molecular technology known as phage display will be used to clone and characterize human anti-Dsg autoantibodies. This technique will allow testing of the following hypotheses: a) mAbs against Dsg1 and Dsg3 from pemphigus patients are both pathogenic and non-pathogenic and both types can be isolated; b) There is genetically restricted heavy and light variable chain usage in anti-Dsg antibodies; c) Pathogenic mAbs bind a restricted set of epitopes at the amino terminus of Dsg3 and Dsg1; d) Pathogenic autoantibody idiotypes are shared among different patients; and e) Pathogenic idiotypes can be blocked by peptides mimicking the Dsg3 or Dsg1 pathogenic epitopes. Antibodies will be cloned from phage display libraries made from patients by selection on Dsg3 and Dsg1. The resulting human mAbs will be characterized by enzyme-linked immunosorbent assay, immunoflourescence and Western blotting. The mAbs will be tested for pathogenicity and then the genetics of the pathogenic and non-pathogenic antibodies will be compared. Phage peptide libraries will be screened with pathogenic mAbs. The respective epitopes on Dsgs will be mapped, and cross-reactive idiotypes (within and among patients) will be defined. The results of these experiments will greatly increase our knowledge of human autoantibodies in pemphigus. The project will: define how different human antibodies contribute to the pathology of this disease, develop valuable human monoclonal antibody reagents for other investigators studying these and other skin diseases, and will point to new ways to address targeted therapy and diagnosis to pathogenic antibodies in these potentially life-threatening diseases.