Immunoregulation of Myelin Specific T Lymphocytes

描述（由申请人提供）： 鉴于现有的治疗方法对疾病进程的影响有限，人类多发性硬化症（MS）需要新的和改进的药物。目前的药物表现出比以前的一线治疗（干扰素-β和醋酸格拉替雷）更好的疗效，但由于全身免疫抑制，大大增加了毒性和严重危及生命的感染风险。优选的治疗方法将是安全且耐受性良好的生物制剂，其具有抑制参与MS发病机制的关键疾病相关机制的有效能力。我们的实验室发现并正在开发部分MHCII类构建体（pMHC）作为MS的可能免疫疗法。与致脑炎髓鞘少突胶质细胞糖蛋白（MOG）-35-55肽共价连接（pDR 2/MOG-35-55）可以逆转CNS炎症和MOG-肽诱导的实验性自身免疫性脑脊髓炎（EAE）的临床体征DR 2转基因小鼠。相同的结构（称为RTL 1000）最近在MS患者的I期安全性试验中成功使用。我们有强有力的临床前证据来支持这样的想法，即RTL 1000可以特异性靶向肽特异性T细胞和对其他致脑炎髓鞘肽反应的旁观者T细胞。pMHC复合物的这种抑制活性需要将pMHC 1 1部分结合到主要在单核细胞上表达的特异性受体上。我们最近发现，pMHC可以特异性地结合到MHC II类（CD 74）的不变链，下调其在单核细胞表面的表达，并阻断巨噬细胞移动抑制因子（MIF）的炎症效应，MIF是EAE和MS的关键致病细胞因子。我们进一步证明，DR-1结构域是pDR 2构建体的主要CD 74结合部分。此外，增加DR-1浓度（在没有结合抗原肽的情况下）可以下调单核细胞上的CD 74水平。这些结果表明，DR-1单独可能通过其对CD 74表达的影响调节MIF活性并以抗原肽非依赖性方式治疗EAE。事实上，我们的初步实验证明了DR-1对EAE的显著治疗效果。这些数据具有重要的调控意义，使得能够开发一类新的治疗性DR-1衍生构建体，用于涉及通过CD 74的MIF信号传导的自身免疫性和炎症性疾病。我们设想DR-κ 1通过其CD 74结合区对CD 74的调节将在广泛的炎性病症中提供显著的治疗效果，即使在致病性T细胞的特异性尚未建立时，并且治疗的功效可以通过将特异性抗原肽附着到DR-κ 1上来增强。1部分在已知或强烈怀疑致病性T细胞特异性的情况下（例如，到MS中的髓磷脂肽）。此外，由于DR-1结构域存在于所有人中，因此不会被识别为外来的，使用DR-1构建体的治疗不需要对潜在受体进行HLA筛查，并且可以用于治疗不表达HLA-DR 2风险因子的MS受试者。我们的初步数据表明EAE小鼠和MS受试者单核细胞上的CD 74水平显著增加，从而为开发通过下调CD 74表达抑制MIF活性的pMHC构建体提供了令人信服的理由。在本申请中，我们将测试DR-1的特定片段与单核细胞结合并降低CD 74和MIF信号传导的细胞表面表达的假设，这反过来又阻止细胞迁移到CNS和Tef细胞活化，从而逆转EAE的临床和组织学体征。为了解决这一假设，我们建议：1）鉴定DR-1内的CD 74结合区域，并开发最佳的CD 74结合DR-1构建体; 2）评价DR-1构建体对单核细胞和T细胞活化的抑制作用; 3）确定DR-1构建体治疗由肽特异性与非特异性（旁观者）T细胞特异性诱导的EAE的功效。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT New and improved drugs are needed for human multiple sclerosis (MS) given that available treatments have only modest effects on the disease course. Current drugs demonstrate better efficacy than the previous first- line treatments (interferon-¿ and glatiramer acetate), but have greatly increased toxicity and risk of serious life- threatening infections due to global immunosuppression. A preferred treatment approach would be a safe and well-tolerated biologic with a potent ability to inhibit key disease-associated mechanisms involved in the pathogenesis of MS. Our laboratory discovered and is developing partial MHC class II constructs (pMHC) as a possible immunotherapy for MS. pMHC containing the extracellular domains of the MS risk factor, HLA-DR2, linked covalently to the encephalitogenic myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide (pDR2/MOG-35-55) can reverse CNS inflammation and clinical signs of MOG-peptide-induced experimental autoimmune encephalomyelitis (EAE) in DR2 transgenic mice. The same construct (termed RTL1000) was recently used successfully in a Phase 1 safety trial in MS patients. We have strong pre-clinical evidence to support the idea that RTL1000 can specifically target both peptide-specific and bystander T-cells reactive to other encephalitogenic myelin peptides. This inhibitory activity of the pMHC complexes requires binding of the ¿1¿1 moiety to a specific receptor expressed predominantly on monocytes. We recently we discovered that pMHC can bind specifically to the invariant chain of MHC class II (CD74), down-regulating its expression on the monocyte cell surface and blocking the inflammatory effects of macrophage migration inhibitory factor (MIF) that is a key pathogenic cytokine for EAE and MS. We further demonstrated that the DR-¿1 domain is the major CD74 binding moiety of pDR2 constructs. Moreover, increased concentrations of DR-¿1 (in the absence of a bound antigenic peptide) could down-modulate CD74 levels on monocytes. These findings suggested that DR-¿1 alone might modulate MIF activity and treat EAE in an antigenic peptide-independent manner through its effects on CD74 expression. Indeed, our preliminary experiments demonstrated a significant treatment effect of DR-¿1on EAE. These data have important regulatory implications that enable the development of a new class of therapeutic DR-¿1-derived constructs for autoimmune and inflammatory conditions involving MIF signaling through CD74. We envision that modulation of CD74 by DR-¿1 through its CD74 binding region will provide significant therapeutic effects in a wide spectrum of inflammatory conditions even when the specificity of pathogenic T-cells has not been established, and that efficacy of treatment may be enhanced by attachment of specific antigenic peptides to the DR-¿1 moiety in conditions where pathogenic T- cell specificities are known or strongly suspected (eg. to myelin peptides in MS). Moreover, because the DR- ¿1 domain is present in all humans and thus would not be recognized as foreign, treatment using DR-¿1 constructs would not require HLA screening of potential recipients and could be used for treatment of MS subjects who do not express the HLA-DR2 risk factor. Our preliminary data suggest a significant increase in CD74 levels on monocytes in mice with EAE and subjects with MS, thus providing a compelling rationale for development off pMHC constructs that can inhibit MIF activity by down-regulating CD74 expression. In this application we will test the hypothesis that a specific segment of DR-¿1 binds to monocytes and decreases cell-surface expression of CD74 and MIF signaling that in turn blocks cell migration into the CNS and Teff-cell activation, resulting in reversal of clinical and histological signs of EAE. To address this hypothesis, we propose to 1) Identify the CD74-binding region within DR-¿1 and develop an optimal CD74-binding DR-¿1 construct; 2) Evaluate inhibitory effects of DR-¿1 constructs on monocyte and T-cell activation; and 3) Determine efficacy of DR-¿1 constructs for treatment of EAE induced by peptide-specific vs. non-specific (bystander) T-cell specificities.