Novel Antigen-Specific Immunomodulatory Tregitope-based Therapy to Address Autoimmune Pathogenesis in Graves' Disease

摘要 意义：Graves病（GD）是一种最常见的自身免疫性疾病， 刺激甲状腺刺激激素受体（TSHR）的自身抗体（TSAb），导致 甲状腺机能亢进目前治疗GD患者甲状腺功能亢进症的方法以过量甲状腺激素为目标 通过切除或切除甲状腺或通过阻断甲状腺激素合成来分泌。这一切成功都 治疗与不良反应有关，最常见的是甲状腺功能减退症，需要终身 替代疗法重要的是，这些方法都没有解决潜在的抗TSHR CD 4 T 细胞反应，有助于持续产生TSAb自身抗体。自身免疫是 由于T效应细胞：T调节细胞（Teff：Treg）免疫细胞稳态失衡，以及中枢免疫功能丧失， 或外周免疫耐受。在IgG中发现的T调节表位（Tregitopes）刺激增殖 并激活天然和诱导的Treg，从而下调Teff对自身抗原的反应。 当与MHC II类结合自身抗原肽一起施用时，这些Tregitopes将引发抗原结合。 特异性适应性耐受诱导（ASATI）。假设：Tregitopes，与靶标组合 抗原-TSHR CD 4 T细胞效应肽-将减少CD 4 T细胞活化和T依赖性B细胞 生产TSAB。由于肽在体内的半衰期短，实际的Tregitope生物治疗剂 （Treg-BT）将需要在改善其PK/PD性质的合适的递送系统中施用 毒性最小。该项目将确定TSHR肽，刺激CD 4效应免疫 在来自GD患者的PBMC中的应答，并且将最佳肽与Tregitopes组合联合收割机，以有效地降低Tregitopes的活性。 送货车在特异性目标1中，TSHR肽被鉴定为刺激CD 4效应细胞应答， GD患者将在TSHR旁观者抑制测定中测试Tregitopes的抑制。Tregitope 然后将具有和不具有TSHR肽的肽配制成两种Treg-BT递送载体： 化学连接到FDA批准的重组人血清白蛋白（HSA）上，或B）配制成 聚乳酸-乙醇酸（PLGA）微球。然后将在TSHR旁观者中测试Treg-BT 使用来自GD患者的PBMC的抑制测定。在具体目标2中，候选Treg-BT（HSA-BT）是一种特异性靶点。 Tregitope + TSHR肽或微球中的Tregitope + TSHR肽，具有适当的对照）将 在GD的小鼠模型中进行体内测试。HLA-DR 3小鼠将用免疫球蛋白进行遗传免疫。 编码TSHR胞外结构域的表达质粒以产生TSAb。TSAb应答后 建立后，将用优化的Treg-BT和血清TSAb和CD 4 T细胞治疗小鼠 将对反应进行监测。总体影响：这些研究将产生候选Treg-BT， 与TSHR肽组合，刺激Treg介导的TSAb自身抗体下调， 潜在的快速通道适用于临床Graves病患者的治疗。

ABSTRACT Significance: Graves’ disease (GD), one of the most prevalent autoimmune diseases, is caused by stimulating autoantibodies (TSAb) to the Thyroid-Stimulating Hormone Receptor (TSHR), resulting in hyperthyroidism. Current methods for managing hyperthyroidism in GD target excess thyroid hormone secretion by ablation or removal of the thyroid or by blocking thyroid hormone synthesis. Each of these therapies is associated with adverse effects, most commonly hypothyroidism requiring life-long replacement therapy. Importantly, none of these approaches address the underlying anti-TSHR CD4 T cell response that contributes to the continued production of TSAb auto-antibodies. Autoimmunity is the result of an imbalance in Teffector:Tregulatory (Teff:Treg) immune-cell homeostasis, and a loss of central or peripheral immune tolerance. T regulatory epitopes (Tregitopes) found in IgG stimulate proliferation and activation of natural and induced Tregs, thereby downregulating a Teff response to self-antigens. These Tregitopes when administered with MHC Class II binding self-antigen peptides, will elicit Antigen- Specific Adaptive Tolerance Induction (ASATI). Hypothesis: Tregitopes, in combination with the target antigen —TSHR CD4 T cell effector peptides — will reduce CD4 T cell activation and T-dependent B cell production of TSAb. Due to the short half-life of peptides in vivo, a practical Tregitope BioTherapeutic (Treg-BT) will need to be administered in a suitable delivery system, that improves its PK/PD properties with minimal toxicity. This project will identify TSHR peptides that stimulate a CD4 effector immune response in PBMC from GD patients, and combine the optimal peptide(s) with Tregitopes in an effective delivery vehicle. In Specific Aim 1, TSHR peptides identified to stimulate a CD4 effector cell response in GD patients will be tested for inhibition by Tregitopes in a TSHR bystander suppression assay. Tregitope peptides with and without TSHR peptides will then be formulated into two Treg-BT delivery vehicles: a) chemically ligated to a recombinant FDA-approved human serum albumin (HSA) or b) formulated into poly(lactic-glycolic acid) (PLGA) microspheres. The Treg-BT will then be tested in the TSHR bystander suppression assay with PBMC from GD patients. In Specific Aim 2, the candidate Treg-BT (HSA- Tregitope + TSHR peptide or Tregitope + TSHR peptides in microspheres, with appropriate controls) will be tested in vivo in a mouse model of GD. HLA-DR3 mice will be genetically immunized with an expression plasmid coding for the extracellular domain of TSHR to generate TSAb. After TSAb response is established, the mice will be treated with the optimized Treg-BT and serum TSAb and CD4 T cell responses will be monitored. Overall Impact: These studies will produce candidate Treg-BT which, combined with TSHR peptides, stimulate a Treg-mediated down-regulation of TSAb autoantibodies, with potential fast-track applicability to the treatment of patients with clinical Graves’ disease.