Molecular, Functional and Structural Analyses of Anti-PAD Antibodies in Rheumatoid Arthritis

项目摘要 风湿性关节炎（RA）是一种常见的慢性炎症性疾病，其导致关节肿胀和疼痛， 放射损伤和残疾抗瓜氨酸蛋白抗体（ACPA）对RA的特异性约为95%。 瓜氨酸酶是精氨酸残基脱亚胺化的翻译后过程，由肽基精氨酸催化 脱亚胺酶（PAD），例如PAD 4。多种蛋白质在RA中被瓜氨酸化，并且PAD 4被认为是 通过提供瓜氨酸化自身抗原的持续来源， 自身免疫反应Darrah博士及其同事鉴定了针对PAD 4本身的抗体（Abs）， 与以下因素相关：（i）疾病严重程度，（ii）血清ACPA，和（iii）严重侵蚀性关节损伤。他们还 发现了与PAD 3交叉反应的抗PAD 4抗体的子集。这些意外导致PAD 4增加 酶活性及其在血清中的存在定义了表现出最侵蚀性的RA患者亚组 损害关于B细胞/抗体对PAD 4的免疫应答， RA中的PAD 3。这些问题将使用表型良好的RA患者的登记/储存库来解决 （主要是高加索人）和访问大型数据库和非裔美国人与RA的知识库。小说 尖端技术将用于研究单个细胞水平的B细胞免疫应答，包括 来自血清的配对重链和轻链抗体和来自循环的转录物的测序 浆母细胞/B细胞。我们将定义抗PAD抗体的分子特征（特异性，表位作图， 等）。结合上述功能研究，将使用结构研究来定义分子结构。 包含与抗-PAD 4或抗-PAD 4/3 mAb（Fab）结合的PAD 4抗原的免疫复合物的决定簇 变体）。因此，抗体介导效应的细胞、分子、生物化学和结构机制 PAD 4将被删除。我们的具体目标是：1。鉴定和定量抗- RA患者血清中PAD IgG和伊加抗体的循环，并确定B细胞亚群的起源。我们 将表征RA患者中的抗PAD 4和PAD 4/3 IgG和伊加抗体蛋白库，并进行 关键B谱系亚群的下一代测序。这将使我们能够识别，量化，并按丰度排名 并将抗PAD库中的所有IG克隆型亚类化。2.确定抗PAD的分子相关物 结合和调节PAD 4活性。a.我们将定义单克隆PAD的分子特征， PAD 4结合、与PAD 3的交叉反应性和PAD 4催化的活化所需的抗体。 利用RA患者血清，我们将直接确定多克隆循环免疫球蛋白的IG类别和亚类库， RA血清中的抗PAD抗体，并确定这些如何与临床特征相关。3.到 确定含PAD的免疫复合物的结构和抗PAD 4/3的机制， 交叉反应性抗体介导PAD 4活化。结构研究将确定的分子决定因素 免疫复合物，其包含与抗-PAD 4或抗-PAD 4/3 mAb（Fab变体）结合的PAD 4抗原。

Project Summary Rheumatoid arthritis (RA) is a common chronic inflammatory disease that results in joint swelling and pain, radiographic damage, and disability. Anti-citrullinated protein antibodies (ACPA) are ~95% specific for RA. Citrullination, a post-translational process of deimination of arginine residues, is catalyzed by peptidylarginine deiminases (PADs) such as PAD4. A variety of proteins are citrullinated in RA, and PAD4 is thought to contribute to RA pathogenesis by providing a continual source of citrullinated autoantigens that induce autoimmune responses. Dr. Darrah and colleagues identified antibodies (Abs) to PAD4 itself, which are associated with: (i) disease severity, (ii) serum ACPA, and (iii) severe erosive joint damage. They also discovered a subset of anti-PAD4 Abs cross-reactive with PAD3. These unexpectedly cause increased PAD4 enzymatic activity and their presence in serum defines a subset of RA patients exhibiting the most erosive damage. There are many unanswered questions regarding B cell/antibody immune responses to PAD4 and PAD3 in RA. These questions will be addressed using a registry/repository of well-phenotyped RA patients (mostly Caucasian) and access to a large database and repository of African-Americans with RA. Novel cutting-edge techniques will be used to study B cell immune responses at the individual cell level, including sequencing of paired heavy and light chain antibodies from serum and transcripts from circulating plasmablasts/B cells. We will define molecular characteristics of anti-PAD Abs (specificity, epitope mapping, etc.). In tandem with the functional studies above, structural studies will be used define the molecular determinants of immune complexes comprising PAD4 antigen bound to anti-PAD4 or anti-PAD4/3 mAbs (Fab variants). Thus, the cellular, molecular, biochemical, and structural mechanisms of antibody-mediated effects on PAD4 will be elucidated. Our Specific Aims are: 1. To identify and quantify the molecular species of anti- PAD IgG and IgA antibodies circulating in serum of RA patients and to identify the B-cell subset of origin. We will characterize anti-PAD4 and PAD4/3 IgG and IgA antibody protein repertoires in RA patients and perform Next-Gen sequencing of key B lineage subsets. This will allow us to identify, quantify, and rank by abundance and subclass all Ig clonotypes in the anti-PAD repertoires. 2. To define the molecular correlates of anti-PAD binding and modulation of PAD4 activity. a. We will define the molecular characteristics of monoclonal PAD antibodies that are required for PAD4 binding, cross-reactivity with PAD3, and activation of PAD4 catalysis. Using serum of RA patients, we will directly define the Ig class and subclass repertoire of polyclonal circulating anti-PAD antibodies in RA serum and determine how these may correlate with clinical characteristics. 3. To determine the structure of PAD-containing immune complexes and the mechanisms by which anti-PAD4/3 cross-reactive antibodies mediate PAD4 activation. Structural studies will define the molecular determinants of immune complexes comprising PAD4 antigen bound to anti-PAD4 or anti-PAD4/3 mAbs (Fab variants).