Thyroid Derived Peptide Presentation by HLA-DR in Thyroiditis

一、家长助学金的研究计划 A)上级赠款摘要 自身免疫性甲状腺疾病(AITD)，包括Graves病(GD)和桥本甲状腺炎 (HT)目前只能对症治疗，而不是通过针对导致 疾病。我们的长期目标是通过阻断来设计针对AITD的精确靶向疗法 将甲状腺抗原呈递给T细胞。为了阻止抗原递送，我们正在 靶向HLA-DRB1-Arg74，我们先前已经证明它是关键的HLA-DR口袋， 呈现可触发AITD的甲状腺多肽。在上一次拨款期间，我们取得了重大进展 朝着我们长期目标的进展：(1)我们确定了引起TSHR的关键多肽表位 Gd；(2)我们鉴定了Cepharanthine是一种阻断DRB1-Arg74并预防AITD的化合物 在小鼠模型中；(3)我们鉴定了阻断DRB1-Arg74结合的D-肽；(4) 利用全基因组方法，我们确定了新的AITD易感基因(例如ARID5B)；(5) 我们确定了触发AITD的遗传-表观遗传交互作用。在取得进展的基础上再接再厉 在之前的赠款期间，我们建议通过以下方式开发AITD的新治疗方法 阻断抗原递送。我们的假设是致病的TG/TSHR的呈现 我们发现DRB1-Arg74口袋中T细胞的多肽是触发AITD的关键，即 与此口袋结合的封闭肽可用于治疗/预防AITD。我们的具体目标是： 具体目标1：我们将提高千金藤碱封闭抗原的效力和疗效 通过创建修饰的Cepharanthine类似物(MCA)在DRB1-Arg74中呈现。我们会 使用计算机方法来设计MCA；MCA将使用我们独特的设计来确认 酶联免疫吸附试验和基于细胞的检测，以及在体外和体内对人源化的DR3的实验 我们将诱发自身免疫性甲状腺炎的小鼠。 具体目标2：我们将产生针对人类白细胞抗原-DRB1-Arg74的单抗 -Tg.2098综合体。这种方法是基于我们的研究结果，即TG肽的呈现 DRB1-Arg74的Tg.2098是触发AITD的关键步骤。我们生产的单抗将是 通过我们的体外ELISA法和基于细胞的分析进行筛选，并证实在我们的 DR3“人源化”AITD小鼠模型。 具体目标3：我们将验证MCA和MAb的阻断抗原提呈在患者中的作用 AITD。我们将通过测试MCA和MAb抑制T细胞召回反应的能力来验证它们的有效性 AITD患者外周血单个核细胞中甲状腺抗原的检测 DRB1-Arg74呈阳性。 总而言之，我们的多学科翻译项目建立在 上一个授权期。我们的目标是致力于AITD新疗法的临床前开发 基于阻断HLA-DRB1-Arg74的抗原提呈。我们的协作团队拥有 能力、经验和专业知识，以实现我们提案的目标。我们的主要优势是 治疗方法是两者都是选择性的，因为只有T细胞识别致病的甲状腺 多肽是有针对性和个性化的，因为只有携带HLA-DRB1-Arg74的患者将 治疗过了。我们的翻译研究有望为AITD带来新的治疗方法。

I. RESEARCH PLAN OF THE PARENT GRANT A) SUMMARY OF THE PARENT GRANT Autoimmune thyroid diseases (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT) are currently treated only symptomatically and not by targeting the mechanisms causing disease. Our long-term goal is to design precision-targeted therapies for AITD by blocking presentation of thyroidal antigens to T-cells. In order to block antigen presentation, we are targeting HLA-DRb1-Arg74, which we have previously shown to be the key HLA-DR pocket that presents thyroidal peptides triggering AITD. During the last grant period we made significant progress towards our long-term goals: (1) We identified the key TSHR peptide epitope causing GD; (2) We identified Cepharanthine as a compound that blocks DRb1-Arg74 and prevents AITD in a mouse model; (3) We identified D-peptides that block peptide binding to DRb1-Arg74; (4) Using genome-wide approaches we identified new AITD susceptibility genes (e.g. ARID5B); (5) We identified genetic-epigenetic interactions triggering AITD. Building on the progress made in the previous grant period we propose to develop new therapeutic approaches for AITD by blocking antigen presentation. Our hypothesis is that presentation of pathogenic Tg/TSHR peptides to T-cells within the DRb1-Arg74 pocket we discovered is key to triggering AITD, & that blocking peptide binding to this pocket can be used to treat/prevent AITD. Our specific aims are: Specific Aim 1: We will enhance the potency and efficacy of Cepharanthine in blocking antigen presentation within DRb1-Arg74 by creating modified Cepharanthine analogs (MCA's). We will use in silico methods to design MCA's; MCA's will be confirmed using our uniquely designed ELISA and cell-based assays, and using ex vivo and in vivo experiments in “humanized” DR3 mice in which we will induce autoimmune thyroiditis. Specific Aim 2: We will generate monoclonal antibodies (MAb's) targeting the HLA-DRb1-Arg74 – Tg.2098 complex. This approach is based on our findings that presentation of the Tg peptide Tg.2098 by DRb1-Arg74 is the key step in triggering AITD. The MAb's we produce will be screened by our in vitro ELISA and cell-based assays, and confirmed ex vivo and in vivo in our DR3 “humanized” mouse model of AITD. Specific Aim 3: We will validate that MCA's & MAb's block antigen presentation in patients with AITD. We will validate MCA's and MAb's by testing their ability to inhibit T cell recall responses to thyroidal antigens in peripheral blood mononuclear cells (PBMC's) isolated from AITD patients that are positive for DRb1-Arg74. In summary, our multidisciplinary translational project builds on the knowledge gained in the previous grant period. Our goal is to pursue preclinical development of novel therapies for AITD based on blocking antigen presentation by HLA-DRb1-Arg74. Our collaborative team has the capacity, experience, & expertise to achieve the aims of our proposal. The main advantage of our therapeutic approach is that it is both selective since only T-cells recognizing pathogenic thyroidal peptides are targeted and personalized since only patients carrying the HLA-DRb1-Arg74 will be treated. Our translational studies will hopefully lead to novel therapies for AITD.