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

类风湿关节炎抗 PAD 抗体的分子、功能和结构分析

基本信息

批准号：
9893071
负责人：
S Louis Bridges
金额：
$ 39.27万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-12 至 2020-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9893071
关键词：
Address Affinity African American Antibodies Antibody Response Antibody Specificity Antigen-Antibody Complex Arginine Arthralgia Autoantibodies Autoantigens Autoimmune Diseases Autoimmune Responses Autoimmunity B-Lymphocyte Subsets B-Lymphocytes Binding Biochemical Biological Process Calcium Catalysis Caucasians Cell Lineage Cells Characteristics Chronic Citrulline Clinical Complex Databases Development Diagnosis Diagnostic radiologic examination Disease Enzymes Epitope Mapping Epitopes Evolution Exhibits Family Family member Generations Histones Immune Tolerance Immune response Immunoglobulin A Immunoglobulin G Individual Infectious Agent Inflammatory Inflammatory Arthritis Lead Light Malignant Neoplasms Mediating Molecular Monoclonal Antibodies Neurodegenerative Disorders Pathogenesis Pathogenicity Pathologic Patients Phenotype Plasmablast Play Post-Translational Protein Processing Process Production Progressive Disease Protein-arginine deiminase Proteins Registries Reporting Research Rheumatoid Arthritis Rheumatoid Factor Role Serum Severity of illness Somatic Mutation Source Stimulus Structure Synovitis Techniques Therapeutic Agents Transcript Variant anti-IgG antigen binding base citrullinated protein cross reactivity dimer disability enzyme activity experimental study improved individual patient inhibitor/antagonist innovation joint inflammation joint injury monomer next generation sequencing novel repository response seropositive structural biology systemic autoimmune disease treatment strategy

项目摘要

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).

项目摘要类风湿关节炎（RA）是一种常见的慢性炎性疾病，导致关节肿胀和疼痛，影像学损害和残疾。抗硝化蛋白抗体（ACPA）对RA的特异性约为95％。柠檬化是精氨酸残基脱丝质的翻译过程，由肽基金氨酸催化脱氨酶（垫），例如PAD4。各种蛋白质在RA中被柑橘粉，Pad4被认为是通过提供持续的柠檬酸自动抗原来源来促进RA发病机理自身免疫反应。 Darrah博士及其同事确定了PAD4本身的抗体（ABS）与：（i）疾病严重程度，（ii）血清ACPA和（iii）严重的侵蚀性关节损伤。他们也是发现了与PAD3的抗Pad4 ABS交叉反应的子集。这些意外导致PAD4增加酶活性及其在血清中的存在定义了一部分RA患者表现出最侵蚀性的患者损害。关于B细胞/抗体免疫对PAD4和 RA中的PAD3。这些问题将使用良好的RA患者的注册表/存储库来解决（主要是高加索人），并使用RA的非裔美国人的大型数据库和存储库。小说尖端技术将用于研究单个细胞水平的B细胞免疫反应，包括从血清和循环中的成对的重链和轻链抗体测序 Plasmablasts/b细胞。我们将定义抗Pad ABS的分子特征（特异性，表位映射， ETC。）。与上述功能研究同时，将使用结构研究定义分子与抗Pad4或抗Pad4/3 mAb结合的PAD4抗原的免疫复合物的决定因素（FAB 变体）。因此，抗体介导的作用的细胞，分子，生化和结构机制在PAD4上将阐明。我们的具体目的是：1。识别和量化抗 - PAD IgG和IgA抗体在RA患者血清中循环并鉴定出原点的B细胞子集。我们将表征RA患者的抗Pad4和Pad4/3 IgG和IgA抗体蛋白质谱并进行关键B谱系子集的下一代测序。这将使我们能够通过丰富来识别，量化和排名和抗Pad曲目中的所有Ig clonotypes。 2。定义抗Pad的分子相关性 PAD4活性的结合和调制。一个。我们将定义单克隆垫的分子特性 PAD4结合，与PAD3的交叉反应性以及PAD4催化的激活所需的抗体。使用RA患者的血清，我们将直接定义多克隆循环的Ig类和亚类曲目 RA血清中的抗PAD抗体，并确定这些抗体与临床特征如何相关。 3 确定含垫的免疫复合物的结构和抗Pad4/3的机制交叉反应抗体介导PAD4激活。结构研究将定义分子决定因素包含与抗Pad4或抗Pad4/3 mAb（FAB变体）结合的PAD4抗原的免疫复合物。