REGULATION OF EAE WITH RECOMBINANT TCR LIGANDS

描述（由申请人提供）：本项目的总体目标是严格检验髓磷脂抗原反应性T细胞和MS风险相关HLA-DR 2等位基因有助于多发性硬化（MS）发病机制的一般假设。为此，我们开发了一种重组TCR配体（RTL），一种单链双结构域人HLA-DR 2 II类分子共价连接到免疫显性MOG-35-55表位，诱导长期耐受性和逆转转基因（Tg）DR 2表达小鼠中MOG肽诱导的EAE的既定临床体征。我们产生的初始DR 2/MOG-35-55构建体（VG 312）具有自粘附表面，并且倾向于形成平均组成为14个亚基的稳定寡聚体。寡聚化对构建体的抑制功能的影响尚不清楚，但潜在地，寡聚体可能具有较低的功能摩尔浓度，因此体内抑制活性低于单体。另一方面，寡聚体可能能够比单体更有效地交联TCR，导致通过TCR的更多或不同的信号传导，这可能影响抑制活性。我们现在已经通过修饰自接触表面中的氨基酸残基产生了DR 2/MOG-35-55单体（VG 342），这将允许与14聚体VG 312构建体进行功能比较。此外，我们已经产生了包含小鼠（m）MOG与人（h）MOG肽的构建体，所述肽对来自患有EAE的小鼠的TCR具有不同的亲和力，以及缺乏凝血酶（T）切割位点的新的单体形式（VG 342-T），所述凝血酶（T）切割位点被工程化到原始构建体的肽连接区中。本申请中我们的目标是比较这五种形式的DR 2/MOG-35-55构建体的治疗功效、耐受性诱导以及对小鼠和人MOG-35-55特异性T细胞系、克隆和我们最近从DR 2小鼠开发的杂交瘤的影响。具体地，在本申请中，我们将阐述耐受性的程度和机制受以下因素支配或影响的特定假设：1）RTL对TCR的亲和力的差异，2）RTL与TCR结合的功能亲合力的差异，和3）游离肽从RTL的切割和释放。最后，我们将评估由RTL诱导的旁观者抑制的程度，并跟踪RTL处理的T细胞的命运。这是首次在体内评价单体与寡聚体TCR阻断对耐受性的影响的研究，并将为这些重组TCR配体（RTL）在MS患者中的临床应用提供必要的基础。

DESCRIPTION (provided by applicant): The overall goal of this program is to test critically the general hypothesis that myelin antigen reactive T cells and the MS risk-associated HLA-DR2 allele contribute to the pathogenesis of multiple sclerosis (MS). To this end we developed a recombinant TCR ligand (RTL), a single chain two domain human HLA-DR2 class II molecule covalently linked to the immunodominant MOG-35-55 epitope that induced long-term tolerance and reversed established clinical signs of MOG-peptide-induced EAE in transgenic (Tg) DR2-expressing mice. The initial DR2/MOG-35-55 construct (VG312) that we produced had self-adherent surfaces and tended to form stable oligomers with an average composition of 14-subunits. The effect of oligomerization on the inhibitory function of the constructs is unknown, but potentially the oligomers might have a lower functional molarity and thus less inhibitory activity in vivo than the monomers. On the other hand, the oligomers might be able to cross-link the TCRs more efficiently than monomers, resulting in more or different signaling through the TCR that might affect inhibitory activity. We have now produced a DR2/MOG-35-55 monomer (VG342) by modifying amino acid residues in the self-contact surface that will allow a functional comparison with the 14-mer VG312 construct. Additionally, we have produced constructs that contain mouse (m)MOG versus human (h)MOG peptides with different affinities for TCRs from mice with EAE, and a new monomeric form (VG342-T) that lacks the thrombin (T) cleavage site that was engineered into the peptide-joining region of the original construct. Our goal in this application is to compare these five forms of the DR2/MOG-35-55 construct for therapeutic efficacy, induction of tolerance, and effects on mouse and human MOG-35-55 specific T cell lines, clones, and a hybridoma that we recently developed from the DR2 mice. Specifically, in this application we will address the specific hypothesis that the degree and mechanism of tolerance is governed or influenced by 1) differences in the affinity of the RTL for the TCR, 2) differences in functional avidity of RTL binding to the TCR, and 3) cleavage and release of free peptide from the RTL. Finally, we will evaluate the degree of bystander suppression induced by RTLs and follow the fate of RTL-treated T cells. These are the first studies ever to evaluate the effect on tolerance of monomeric versus oligomeric TCR blockage in vivo, and will provide the necessary foundation for clinical application of these recombinant TCR ligands (RTLs) in patients with MS.