B-CELL RESPONSES TO CLOTTING FACTOR VIII

描述（由申请人提供）：作为致病性 B 细胞反应，因子 VIII 抑制剂代表了主要的临床挑战，包括血友病 A 患者的同种抗体或获得性血友病的自身抗体。反应是针对不同域上有限数量的主要表位的多克隆反应，并且 IgG4 反应通常占主导地位。然而，大多数表征因子 VIII 抑制剂的研究都是针对代表单个时间点的样本，重点关注峰值响应时的高滴度抗体，并定义与整个域中的表位反应的分数。我们建议研究针对因子VIII的C结构域上的表面表位簇的多克隆反应的部分，以表征受试者之间的变异性和个体患者随时间的稳定性（或变化）。我们将研究三组​​患者组：(1) 严重血友病 A 中的同种抗体，(2) 获得性血友病 A 中的自身抗体和 (3) 轻度血友病 A 患者中的自身抗体和同种抗体组合。C 结构域表位仍然是最复杂且最不为人所知的。将表征与 C 结构域表位簇结合的亲和纯化抗体级分。我们已经表达了 C2 和 C1C2 以及几个突变体。将评估与 C2、其突变体或 C1C2 反应的抑制和/或结合患者抗体的相对量以及 IgG 同种型的分布，无论是在患者之间还是在给定患者样本中的纵向分布。特定的血友病 C1C2 突变体将表征轻重度患者对抑制剂的反应。作为最后一个组成部分，我们将定义 C1C2 表面的结构元件，以及 C2 上残基构象的灵活性程度，当整个 C 结构域存在时，表面残基会有所不同。结果将为治疗出血事件和诱导耐受或预防同种免疫的策略提供见解。

DESCRIPTION (provided by applicant): As a pathogenic B cell response, factor VIII inhibitors represent a major clinical challenge including alloantibodies in hemophilia A patients or autoantibodies in acquired hemophilia. Responses are polyclonal to a limited number of major epitopes on different domains and IgG4 responses often predominate. However, most studies to characterize factor VIII inhibitors have been on samples representing single points in time, focusing upon high titer antibodies at their peak response and defining fractions reacting with epitopes throughout entire domains. We propose to study fractions of the polyclonal responses that are directed to clusters of surface epitopes on factor VIIl's C domains to characterize variability between subjects and stability (or changes) across time in individual patients. We will study three sets of patient groups: (1) allo antibodies in severe hemophilia A, (2) auto antibodies in acquired hemophilia A and (3) combined auto and allo antibodies in patients with mild hemophilia A. C domain epitopes remain the most complex and least well understood. Affinity-purified antibody fractions binding to C domain epitope clusters will be characterized. We have expressed C2 and C1C2 and several mutants. Relative amounts of inhibiting and/or binding patient antibodies reacting to C2, its mutants or C1C2 will be assessed as will distribution of IgG isotypes, both between patients and longitudinally in a given patient's samples. Specific hemophilic C1C2 mutants will characterize responses in mildly severe patients with inhibitors. As a final component, we will define structural elements of C1C2's surface, and the extent of flexibility in conformations of residues on C2, surface residues that differ when the entire C domain is present. Results will provide insights into strategies for therapy of bleeding episodes and the induction of tolerance or prevention of alloimmunization.