Cloning and genetics of human pemphigus autoantibodies

描述（由申请人提供）：寻常型天疱疮和叶状天疱疮是严重的，有时危及生命的自身免疫性水泡性皮肤病，其中针对桥粒蛋白（Dsg） 3和1的自身抗体，桥粒体粘附分子，导致角化细胞细胞粘附丧失。最终，更具体的治疗和诊断方法将需要更详细地表征致病自身抗体。通过确定天疱疮患者的个体，即单克隆抗体（mab）的遗传特性和精细特异性，可以解决有关患者之间共同的免疫球蛋白可变段基因使用以及共同的致病独特型和表位的问题。一种被称为噬菌体展示的强大分子技术将用于克隆和表征人类抗dsg自身抗体。该技术将允许测试以下假设：a)针对天疱疮患者Dsg1和Dsg3的单克隆抗体既有致病性也有非致病性，并且两种类型都可以分离；b)抗dsg抗体的重、轻可变链使用存在遗传限制；c)致病性单抗在Dsg3和Dsg1的氨基末端结合一组限制性表位；d)不同患者具有相同的病原性自身抗体独特型；e)病原独特型可以被模拟Dsg3或Dsg1病原表位的肽阻断。抗体将通过选择Dsg3和Dsg1从患者噬菌体展示文库中克隆出来。由此产生的人单抗将通过酶联免疫吸附试验、免疫荧光和Western blotting进行表征。对单克隆抗体进行致病性检测，然后对致病性抗体和非致病性抗体进行遗传比较。噬菌体肽文库将用致病性单抗进行筛选。将绘制Dsgs上各自的表位，并定义交叉反应独特型（患者内部和患者之间）。这些实验的结果将大大增加我们对天疱疮人类自身抗体的认识。该项目将：定义不同的人类抗体如何促进这种疾病的病理，为研究这些和其他皮肤疾病的其他研究人员开发有价值的人类单克隆抗体试剂，并将指出针对这些潜在威胁生命的疾病的靶向治疗和病原抗体诊断的新方法。

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.