Thyroid Derived Peptide Presentation by HLA-DR in Thyroiditis

I.专利补助金的研究 A）专利补助金概要 自身免疫性甲状腺疾病（AITD），包括Graves病（GD）和桥本甲状腺炎 (HT)目前只进行了治疗，而不是针对机制， 疾病我们的长期目标是设计精确的AITD靶向治疗方法， 将甲状腺抗原呈递给T细胞。为了阻断抗原呈递，我们 靶向HLA-DRb 1-Arg 74，我们以前已经证明这是关键的HLA-DR口袋， 甲状腺肽引发AITD。在上一个赠款期间，我们取得了重大进展， 朝着我们的长期目标取得了进展：（1）我们确定了关键的TSHR肽表位， GD;（2）我们鉴定了千金藤素作为阻断DRb 1-Arg 74和预防AITD的化合物 在小鼠模型中;（3）我们鉴定了阻断肽与DRb 1-Arg 74结合的D-肽;（4） 使用全基因组方法，我们鉴定了新的AITD易感基因（例如ARID 5 B）;（5） 我们确定了触发AITD的遗传-表观遗传相互作用。在取得进展的基础上， 在上一个资助期，我们建议通过以下方式开发新的AITD治疗方法： 阻断抗原呈递。我们的假设是，致病性Tg/TSHR 我们发现，在DRb 1-Arg 74口袋内的T细胞肽是触发AITD的关键， 阻断肽与该口袋的结合可用于治疗/预防AITD。我们的具体目标是： 具体目标1：提高千金藤素的封闭抗原的效力和功效 通过产生修饰的千金藤素类似物（MCA）在DRb 1-Arg 74内呈递。我们将 使用计算机模拟方法设计MCA; MCA将使用我们独特设计的 ELISA和基于细胞的测定，以及在“人源化”DR 3中使用离体和体内实验 诱发自身免疫性甲状腺炎的小鼠。 具体目标2：我们将产生靶向HLA-DRb 1-Arg 74的单克隆抗体（MAb） - TG.2098复合体。这种方法是基于我们的发现，Tg肽的呈递 Tg.2098通过DRb 1-Arg 74是触发AITD的关键步骤。我们生产的单克隆抗体 通过我们的体外ELISA和基于细胞的测定筛选，并在我们的体外和体内实验中证实， AITD的DR 3“人源化”小鼠模型。 具体目标3：我们将验证MCA和MAb阻断患有以下疾病的患者中的抗原呈递： AITD。我们将通过测试MCA和MAb抑制T细胞回忆反应的能力来验证它们 从AITD患者分离的外周血单个核细胞（PBMC）中的甲状腺抗原 都是DRb 1-Arg 74阳性 总之，我们的多学科翻译项目建立在 上一个补助期。我们的目标是追求临床前开发的新疗法为AITD 基于阻断HLA-DRb 1-Arg 74的抗原呈递。我们的合作团队拥有 能力，经验和专业知识，以实现我们的建议的目标。我们的主要优势 治疗方法是，它是选择性的，因为只有T细胞识别致病性甲状腺肿， 因为只有携带HLA-DRb 1-Arg 74的患者才能被靶向和个性化， 治疗。我们的转化研究将有望为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.