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影响皮肤和粘膜的毁容和潜在威胁生命的自身免疫性起泡性疾病，由抗角质形成细胞粘附分子（称为桥粒蛋白）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。正常人不产生自身抗体（即对正常存在和暴露在体内的分子具有“耐受性”）。天疱疮的发生是因为产生抗体的B细胞对粘连蛋白失去了耐受性，从而产生了导致疾病的抗粘连蛋白抗体。我们将解决与天疱疮病理生理学相关的一个基本问题，即，是否在每个患者中一次性触发导致一组对粘粒蛋白失去耐受性的寡克隆B细胞，并且在整个疾病过程中自身抗体来源于这组无耐受性的亲本B细胞。或者，患有这种疾病的患者是否有一种倾向，即B细胞对粘连蛋白的耐受性丧失，这种抗体来自于不断进化的亲代非耐受性B细胞的多种克隆无关的抗粘连蛋白抗体？我们将通过使用抗体噬菌体展示来解决这些问题，这是一种从患者身上克隆单克隆抗体的方法，这样编码每个抗体的DNA就可以被测序，从而可以对每个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征单克隆抗体从天疱疮患者时，活跃和复发后的临床缓解。利妥昔单抗是一种暂时破坏B细胞，但B细胞重新繁殖的治疗方法，我们将确定患者在长期缓解后是否仍有抗粘粒蛋白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抗粘粒蛋白B细胞克隆，或者复发是否导致与原始克隆无关的抗粘粒蛋白B细胞克隆。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少数抗粘粒蛋白B细胞克隆逃脱耐受性，如果消除（例如通过利妥昔单抗），可能治愈疾病，而在后一种情况下，患者将有失去耐受性的倾向，这种情况更加难以理解和治疗。最后，我们将确定利妥昔单抗治疗是否消除了所有可检测到的抗粘蛋白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自身免疫反应的病理生理学及其治疗意义。

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. PUBLIC HEALTH RELEVANCE: Pemphigus is a disfiguring and potentially fatal blistering autoimmune disease. This project will clone the autoantibodies from these patients to determine if autoimmunity is triggered by a onetime insult or is caused by a basic defect in the patient's ability to regulate their immune system that causes continually evolving sets of autoantibodies. If the former, then therapies that destroy the B cells causing the abnormal antibodies could cure disease.