Cloning and genetics of human pemphigus autoantibodies

描述（由申请人提供）：寻常天疱疮和落叶型天疱疮是严重的、有时危及生命的自身免疫性起泡皮肤病，其中抗桥粒糖蛋白（Dsg）3和1（桥粒粘附分子）的自身抗体导致角质形成细胞粘附丧失。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的单个（即单克隆）抗体（mAb）的遗传特性和精细特异性，可以解决关于常见免疫球蛋白可变区段基因使用以及患者之间共享的致病性独特型和表位的问题。一种强大的分子技术称为噬菌体展示将用于克隆和表征人抗Dsg自身抗体。该技术将允许测试以下假设：a）来自天疱疮患者的抗Dsg 1和Dsg 3的mAb是致病性的和非致病性的，并且两种类型都可以分离; B）在抗Dsg抗体中存在遗传限制的重链和轻链可变链使用; c）致病性mAb在Dsg 3和Dsg 1的氨基末端结合一组限制的表位; d）致病性自身抗体独特型在不同患者之间共享;和e）致病性独特型可以被模拟Dsg 3或Dsg 1致病性表位的肽阻断。通过在Dsg 3和Dsg 1上选择，从由患者制备的噬菌体展示文库克隆抗体。将通过酶联免疫吸附测定、免疫荧光和Western印迹法表征所得人mAb。将检测mAb的致病性，然后比较致病性和非致病性抗体的遗传学。将用致病性mAb筛选噬菌体肽文库。将绘制Dsgs上的相应表位，并定义交叉反应性独特型（患者内和患者间）。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认识。该项目将：确定不同的人类抗体如何促进这种疾病的病理学，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克隆抗体试剂，并将指出新的方法来解决这些潜在威胁生命的疾病中的病原性抗体的靶向治疗和诊断。

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.