Thyroid Derived Peptide Presentation by HLA-DR in Thyroiditis

自身免疫性甲状腺疾病(AITD)，包括Graves病(GD)和桥本甲状腺炎(HT) 目前只针对症状进行治疗，而不是针对致病机制。我们的长期合作 目标是通过阻断甲状腺抗原呈递给T细胞来设计针对AITD的精确靶向治疗。 为了阻止抗原递送，我们将目标对准了HLA-DRB1-Arg74，我们之前已经向 成为触发AITD的甲状腺多肽的关键的HLA-DR口袋。在上一次授权期内，我们 在实现我们的长期目标方面取得了重大进展：(1)我们确定了关键的TSHR多肽表位 (2)我们鉴定千金藤碱是一种阻断DRB1-Arg74并预防AITD的化合物。 小鼠模型；(3)我们鉴定了阻断DRB1-Arg74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(如ARID5B)；(5)我们鉴定了遗传-表观遗传学 引发AITD的相互作用。在前一个授权期取得的进展的基础上，我们建议制定 通过阻断抗原提呈治疗AITD的新方法。我们的假设是 我们发现的致病TG/TSHR多肽对DRB1-Arg74口袋内的T细胞是触发的关键 AITD&结合到这个口袋上的封闭肽可以用于治疗/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在阻断抗原提呈方面的效力和疗效 在DRB1-Arg74中，通过产生修饰的千金藤碱类似物(MCA)。我们将在硅胶中使用方法来 设计MCA；MCA将使用我们独特设计的ELISA法和基于细胞的分析进行确认，并使用 体外和体内实验，在人源化的DR3小鼠，我们将在其中诱导自身免疫性甲状腺炎。 具体目标2：我们将制备针对人类白细胞抗原-DRB1-Arg74-Tg.2098的单抗 很复杂。这种方法是基于我们的发现，即由DRB1-Arg74呈现TG肽Tg.2098是 触发AITD的关键一步。我们生产的单抗将通过我们的体外酶联免疫吸附试验和基于细胞的筛选 在我们的DR3人源化的AITD小鼠模型中进行了体外和体内的检测和确认。 具体目标3：我们将验证MCA和MAb在AITD患者中的阻断抗原提呈。我们会 通过测试MCA和MAb抑制T细胞对甲状腺抗原的召回反应的能力来验证MCA和Mab DRB1-Arg74阳性的AITD患者外周血单个核细胞(PBMC)。 总而言之，我们的多学科翻译项目建立在上一次拨款中获得的知识的基础上 句号。我们的目标是致力于基于封闭抗原的AITD新疗法的临床前开发 由人类白细胞抗原DRB1-Arg74呈递。我们的协作团队拥有实现以下目标的能力、经验和专业知识 我们提案的目的。我们的治疗方法的主要优点是它既是选择性的，因为只有 识别致病甲状腺多肽的T细胞是有针对性的和个性化的，因为只有患者携带 将对HLA-DRB1-Arg74进行治疗。我们的翻译研究有望为AITD带来新的治疗方法。

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.