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

摘要 意义：Graves病(GD)是最常见的自身免疫性疾病之一，由 刺激性促甲状腺激素受体自身抗体(TSAb)，导致 甲亢。GD靶向甲状腺激素过多的甲状腺功能亢进症的现行治疗方法 通过切除或切除甲状腺或阻止甲状腺激素合成而产生的分泌。这其中的每一个 治疗与副作用有关，最常见的是需要终生的甲状腺功能减退症。 替代疗法。重要的是，这些方法都不能解决潜在的抗TSHR CD4T 有助于持续产生TSAb自身抗体的细胞反应。自身免疫力是 T效应器失衡的结果：T调节性免疫细胞稳态和中枢神经系统功能的丧失 或外周免疫耐受性。免疫球蛋白中发现的T调节表位刺激增殖 以及激活自然和诱导的Tregs，从而下调对自身抗原的TJeff反应。 当这些三个表位与MHC II类结合的自身抗原肽一起注射时，将诱导抗原- 特异性适应性耐受诱导(ASATI)。假设：Tregitope，结合目标 抗原-TSHR CD4T细胞效应肽-将减少CD4T细胞活化和T依赖的B细胞 TSAB的制作。由于多肽在体内的半衰期很短，一种实用的Tregitope生物疗法 (Treg-BT)将需要在适当的输送系统中给予，以改善其PK/PD性能 毒性最小。该项目将识别刺激CD4效应免疫的TSHR多肽 GD患者外周血单核细胞的应答，并有效结合最佳多肽(S)和三倍体 送货车辆。在特定目标1中，TSHR多肽被确定为刺激小鼠体内的CD4效应细胞反应 在TSHR旁观者抑制试验中，将测试Tregitope对GD患者的抑制作用。Tregitope 然后，含有和不含有TSHR多肽的多肽将被配方成两种Treg-Bt递送载体：a) 化学连接到FDA批准的重组人血清白蛋白(HSA)或b)，形成 聚乳酸-乙醇酸(PLGA)微球。Treg-BT随后将在TSHR旁观者中进行测试 用GD患者的PBMC进行抑制试验。在具体目标2中，候选Treg-BT(HSA- 微球中的Tregitope TSHR多肽或Tregitope TSHR多肽，并进行适当的对照) 在GD小鼠模型上进行活体试验。人类白细胞抗原-DR3小鼠将获得基因免疫 编码TSHR胞外区的表达载体产生TSAb。在TSAB响应之后 一旦建立，将用优化的Treg-BT和血清TSAb和CD4T细胞治疗小鼠 我们将密切关注他们的回应。总体影响：这些研究将产生候选的Treg-BT， 与TSHR多肽结合，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.