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影响皮肤和粘膜的毁容和潜在危及生命的自身免疫性起泡疾病，由抗角质形成细胞粘附分子（称为桥粒芯糖蛋白）的自身抗体介导。致病性抗体通过干扰细胞粘附直接引起水疱形成。正常个体不产生自身抗体（即对通常存在并暴露于其体内的分子具有“耐受性”）。天疱疮的发生是因为产生抗体的B细胞失去对桥粒芯糖蛋白的耐受性，从而允许它们产生引起疾病的抗桥粒芯糖蛋白抗体。我们将解决一个基本的问题，有关天疱疮的病理生理学，即，是否一次性触发在每个病人导致一组寡克隆B细胞已失去耐受性桥粒芯糖蛋白，和自身抗体在整个疾病过程中来自这一亲本组的非耐受性B细胞。或者，患有疾病的患者是否倾向于丧失B细胞对桥粒芯糖蛋白的耐受性，而多种克隆无关的抗桥粒芯糖蛋白抗体来源于不断进化的亲本非耐受性B细胞组？ 我们将通过使用抗体噬菌体展示来解决这些问题，抗体噬菌体展示是一种从患者克隆单克隆抗体的方法，以便可以对编码每种抗体的DNA进行测序，从而允许对每种克隆抗体进行遗传表征以确定其亲本B细胞克隆来源。我们将纵向克隆和遗传特性天疱疮患者的单克隆抗体时，活动和复发后的临床缓解。我们将确定利妥昔单抗（一种暂时破坏B细胞，但其中B细胞重新增殖的疗法）治疗后长期缓解的患者是否仍有任何抗桥粒芯糖蛋白B细胞克隆。 这些研究将确定单个天疱疮患者是否在整个疾病过程中保持相同的克隆相关的抗桥粒芯糖蛋白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具有重要意义;在前一种情况下，最初的损伤可能导致一些抗桥粒芯糖蛋白B细胞克隆脱离耐受，如果被消除，（例如通过利妥昔单抗），可以治愈疾病，而在后一种情况下，患者将具有失去耐受性的倾向，这种情况更难以理解和治疗。我们将确定利妥昔单抗治疗是否消除了所有可检测的抗桥粒芯糖蛋白B细胞克隆，或者另一方面，以某种方式允许它们的调节，使它们不产生致病性抗体。这些研究将阐明天疱疮的自身免疫反应的病理生理学与治疗的影响。

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.