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影响皮肤和粘膜的毁容并可能危及生命的自身免疫性水泡性疾病，由抗角质形成细胞黏附分子的自身抗体介导，称为桥连蛋白。致病抗体通过干扰细胞黏附直接导致水泡的形成。正常人不会产生自身抗体(即对体内正常存在和暴露的分子具有“耐受性”)。天疱疮的发生是因为产生抗体的B细胞对桥粒蛋白失去耐受性，从而允许它们产生抗桥粒糖蛋白抗体而导致疾病。我们将解决与天疱疮病理生理学有关的一个基本问题，即每个患者的一次性触发是否会导致一组对桥粒蛋白失去耐受性的寡克隆B细胞，以及整个病程中的自身抗体是否来自这组不耐受的亲代B细胞。或者，疾病患者是否有发生B细胞对桥粒芯糖蛋白耐受性丧失的倾向，这些桥粒糖蛋白抗体来源于不断进化的亲代非耐受B细胞集，而多个克隆性无关的抗桥粒糖蛋白抗体？我们将通过使用抗体噬菌体展示来解决这些问题，这是一种克隆患者单抗的方法，以便对编码每个抗体的DNA进行测序，从而使每个克隆抗体的基因特征能够确定其亲代B细胞克隆来源。我们将纵向克隆天疱疮患者的单抗，并对其进行遗传学特征分析，这些抗体来自活跃的天疱疮患者和临床缓解后复发患者。我们将确定在利妥昔单抗治疗后长期缓解的患者是否仍有抗桥粒芯糖蛋白B细胞克隆。利妥昔单抗是一种暂时摧毁B细胞的疗法，但在那些B细胞重新填充的患者中。这些研究将确定天疱疮患者是否在整个疾病过程中保持相同的克隆性相关抗桥粒芯糖蛋白B细胞克隆，或者复发是否导致与原始克隆无关的抗桥粒芯糖蛋白B细胞。这些发现对病理生理学和治疗有重要意义；在前一种情况下，最初的侮辱可能导致少数抗桥粒芯糖蛋白B细胞克隆逃脱耐受，如果消除(例如使用利妥昔单抗)，可能会治愈疾病，而在后一种情况下，患者将有失去耐受性的倾向，这种情况更难理解和治疗。最后，我们将确定利妥昔单抗疗法是否确实消除了所有可检测到的抗桥粒芯糖蛋白B细胞克隆，或者，另一方面，以某种方式允许它们进行调节，使它们不产生致病抗体。这些研究将阐明天疱疮自身免疫反应的病理生理学，并为治疗提供参考。

DESCRIPTION (provided by applicant): Pemphigus vulgaris and foliaceus are disfiguring and potentially life-threatening autoimmune blistering diseases affecting skin and mucous membranes that are mediated by autoantibodies against keratinocyte cell adhesion molecules called desmogleins. The pathogenic antibodies directly cause blister formation by interfering with cell adhesion. Normal individuals do not produce autoantibodies (i.e. are "tolerant" to molecules normally present and exposed in their bodies). Pemphigus occurs because B cells, which produce antibodies, lose tolerance to desmogleins permitting them to produce anti-desmoglein antibodies that cause disease. We will address a basic issue pertaining to the pathophysiology of pemphigus, namely, whether a onetime trigger in each patient causes one set of oligoclonal B cells that have lost tolerance to desmogleins, and autoantibodies throughout the course of disease are derived from this parental set of non-tolerant B cells. Alternatively, do patients with disease have a propensity for developing loss of B cell tolerance to desmogleins with multiple clonally unrelated anti- desmoglein antibodies derived from continually evolving sets of parental non-tolerant B cells? We will address these questions by using antibody phage display, a method to clone monoclonal antibodies from patients so that the DNA encoding each antibody can be sequenced, allowing genetic characterization of each cloned antibody to determine its parental B cell clonal origin. We will longitudinally clone and genetically characterize monoclonal antibodies from pemphigus patients when active and with relapse after a clinical remission. We will determine if patients in long term remission after rituximab, a therapy that destroys B cells temporarily, but in whom the B cells repopulate, still have any anti-desmoglein B cell clones. These studies will determine if individual pemphigus patients maintain the same clonally-related anti-desmoglein B cell clones throughout disease or if relapse results in anti-desmoglein B cells unrelated to the original clones. These findings have important implications for pathophysiology and therapy; in the former case an initial insult may result in a few anti-desmoglein B cell clones that have escaped from tolerance and, if eliminated (e.g. by rituximab), may cure disease, whereas in the later case patients would have a propensity to lose tolerance, a condition much more difficult to understand and treat. Finally, we will determine if rituximab therapy does eliminate all detectable anti-desmoglein B cell clones or, on the other hand, somehow allows their regulation so they do not produce pathogenic antibodies. These studies will elucidate the pathophysiology of the autoimmune response in pemphigus with implications for therapy.