Anti-Axl and anti-Mer antibody treatment to reduce the clinical course of EAE

抗 Axl 和抗 Mer 抗体治疗可减少 EAE 的临床病程

• 批准号: 9222052
• 负责人: BRIDGET SHAFIT-ZAGARDO
• 金额: $ 19.1万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9222052
• 关键词: Affinity; Alzheimer' s Disease; Anti-Inflammatory Agents; Anti-inflammatory; Antibodies; Antibody Activation; Apoptotic; Autoimmune encephalitis; Back; Brain; Cell Surface Receptors; Cell surface; Chronic; Circadian Rhythms; Clinical; Cuprizone; Data; Demyelinating Diseases; Detection; Disease; Environment; Family; Growth; Half-Life; Homeostasis; Human; Immunoglobulin G; Individual; Inflammation; Inflammatory; Injection of therapeutic agent; Intraperitoneal Injections; Intravenous; Length; Lesion; Ligands; Liver; Lung; Maintenance; Manuscripts; Membrane; Modeling; Multiple Sclerosis; Multiple Sclerosis Lesions; Mus; Neurodegenerative Disorders; Organ; Phagocytosis; Phosphotransferases; Proteins; Publishing; Receptor Protein-Tyrosine Kinases; Recovery; Regulation; Reporting; Resolution; Role; Route; Serum; Signal Transduction; Signaling Molecule; Small Intestines; Specificity; Spleen; Stimulus; Testing; Therapeutic; Tissue Model; Tissues; Toxic effect; Wild Type Mouse; axl receptor tyrosine kinase; axon injury; cell type; clinical predictors; cytokine; high risk; in vivo; macrophage; member; mouse model; multiple sclerosis patient; neuroprotection; oligodendrocyte-myelin glycoprotein; public health relevance; receptor; research and development

 DESCRIPTION (provided by applicant): Studies by others, our published and ongoing studies in human and MS tissue and mouse models have characterized the importance of growth arrest-specific protein 6 (Gas6) signaling and members of the Tyro3, Axl, and Mertk (TAM) family of receptor tyrosine kinases (RTKs) during inflammatory demyelinating disease and recovery (1-21,41). The maintenance of a Gas6 reservoir in multiple cell types and organs is dependent on the simultaneous expression on the cell surface of receptor tyrosine kinase Axl demonstrated to aid in the resolution of inflammation in tissue (12,18). Immunohistochemical detection of Gas6 in multiple tissues sections including spleen, small intestine, liver and lung from wild-type (WT), Mertk−/− and Tyro3−/− mice was positive. By contrast, Gas6 was lost in these same tissues from Axl-/− mice, and Gas6 did not accumulate in serum. As Gas6 is the sole ligand for Axl, the data suggest that Axl and Gas6 are co-dependent (18). This is a unique role for a ligand/receptor interaction, and thus is integral for understanding Gas6/Axl downstream signaling. Shown in Figure 1 is a model proposed by Zagorska et al. (18) in which following proinflammatory stimuli, Axl is increased resulting in Axl-dependent phagocytosis and resolution of inflammation that results in shedding of Axl from the cell surface (soluble Axl). They further suggest that Mertk is more involved in homeostatic phagocytosis of circadian tolerogenic macrophages during the maintenance of tissue homeostasis. In vivo intravenous (IV) injection of activating anti-Axl to mice found spleen Axl kinase activity was activated 15 min later, with Axl activity back to baseline 24h later; no change in Mertk was detected. IgG had no effect activating Axl or Mertk. Similar results were observed with a Mertk activating antibody (R&D) with activated Mertk detected after 1h in vivo; however, Mertk remained at the membrane and no soluble Mertk was detected (18). Our findings (7,11,12) support and validate the proposed Model. The rationale for studying the beneficial effect of Axl and Mertk antibodies administered during myelin oligodendrocyte glycoprotein (MOG)- induced experimental autoimmune encephalopathy (EAE) is due to their longer half-life in vivo versus the short half-life of Gas6. Even with its short half-life we observe beneficial effects of icv administration of Gas6 during cuprizone toxicity and MOG-induced (11, 41, see enclosed manuscript). Whether in vivo treatment with activating Axl antibody or Mertk antibody can be anti-inflammatory and beneficial during EAE aiding in the clearance of apoptotic debris and protective to the CNS will be a focus of this study. There is evidence for antibodies crossing the BBB in individuals with neurodegenerative diseases including Alzheimer's disease (22-26). Figure 1: Model for proposed differences in regulation and action of Axl and Mertk (Mer in figure) in inflammatory and homeostatic environments respectively (18). Specific Aim 1 will determine the best route of antibody administration using IP and icv injections to administer either IgG or activating anti-Axl antibody during MOG-induced EAE and PLP-induced EAE to WT mice. We predict that WT mice (n=5/group) receiving anti-Axl antibody will have a less severe disease course with reduced proinflammatory cytokines and fewer axonal spheroids than IgG. We will demonstrate specificity using Axl-/- mice and predict the clinical course will be worse than WT mice treated with IgG and thus not be beneficial. Specific Aim 2 will determine whether prior to the onset clinical scores a single IP injection of activating or inhibiting Mertk antibody can beneficially ater the EAE clinical course relative to IgG and PBS, and maintain the reduced clinical scores and preserve axonal integrity that is lost during chronic EAE. These studies will further characterize the role of Axl and Mertk during EAE using activating antibodies as a possible therapeutic approach to target proinflammatory signaling molecules. This more refined approach will target two of the TAMs since Gas6 activates all three TAMs with a relative affinity of Gas6 for its receptors Axl>Tyro3>Mertk. We will not study Tyro3 as there is no evidence Tyro3 changes during inflammation, EAE or in MS lesions. This more focused approach may provide neuroprotection against axonal damage and loss found in chronic MS CNS tissue, and perhaps is beneficial as a therapy for individuals with progressive MS.

 描述（由申请人提供）：其他人的研究、我们在人类和MS组织和小鼠模型中已发表和正在进行的研究已经表征了生长停滞特异性蛋白6（Gas 6）信号传导和受体酪氨酸激酶（RTK）的Tyro 3、Axl和Mertk（TAM）家族成员在炎性脱髓鞘疾病和恢复期间的重要性（1- 21，41）。在多种细胞类型和器官中Gas 6储库的维持依赖于受体酪氨酸激酶Axl在细胞表面上的同时表达，所述受体酪氨酸激酶Axl被证明有助于组织中炎症的消退（12，18）。在野生型（WT）、Mertk−/−和Tyro 3 −/−小鼠的脾脏、小肠、肝脏和肺等多个组织切片中，Gas 6的免疫组织化学检测呈阳性。相比之下，Gas 6在 这些来自Axl-/-小鼠的相同组织，并且Gas 6不在血清中积累。由于Gas 6是Axl的唯一配体，因此数据表明Axl和Gas 6是共依赖的（18）。这是配体/受体相互作用的独特作用，因此对于理解Gas 6/Axl下游信号传导是不可或缺的。图1显示了Zagorska等人（18）提出的模型，其中在促炎刺激后，Axl增加，导致Axl依赖性吞噬作用和炎症消退，导致Axl从细胞表面脱落（可溶性Axl）。他们进一步表明，Mertk在维持组织稳态期间更多地参与昼夜耐受原性巨噬细胞的稳态吞噬作用。在体内静脉内（IV）注射活化性抗Axl至小鼠发现脾Axl激酶活性在15分钟后被活化，Axl活性在24小时后回到基线;未检测到Mertk的变化。IgG对激活Axl或Mertk没有影响。用Mertk活化抗体（R&D）观察到类似的结果，在体内1小时后检测到活化的Mertk;然而，Mertk保留在膜上，未检测到可溶性Mertk（18）。我们的研究结果（7，11，12）支持并验证了所提出的模型。研究在髓鞘少突胶质细胞糖蛋白（MOG）诱导的实验性自身免疫性脑病（EAE）期间施用的Axl和Mertk抗体的有益作用的基本原理是由于它们在体内的半衰期较长，而Gas 6的半衰期较短。即使其半衰期较短，我们也观察到在铜腙毒性和MOG诱导期间icv施用Gas 6的有益效果（11，41，参见所附手稿）。用活化Axl抗体或Mertk抗体的体内治疗是否可以在EAE期间抗炎和有益，帮助清除凋亡碎片并保护CNS将是本研究的焦点。有证据表明，在患有包括阿尔茨海默病在内的神经退行性疾病的个体中，抗体穿过BBB（22-26）。图一：分别在炎症和稳态环境中Axl和Mertk（图中的Mer）的调节和作用的差异模型（18）。具体目标1将确定使用IP和icv注射来施用IgG或活化抗Axl的抗体施用的最佳途径 在MOG诱导的EAE和PLP诱导的EAE期间，向WT小鼠施用抗体。我们预测，接受抗Axl抗体的WT小鼠（n=5/组）将具有较不严重的疾病过程，具有减少的促炎细胞因子和比IgG更少的轴突球状体。我们将使用Axl-/-小鼠证明特异性，并预测临床过程将比用IgG处理的WT小鼠更差，因此不是有益的。具体目标2将确定在发作临床评分之前，活化或抑制Mertk抗体的单次IP注射相对于IgG和PBS是否可以有益地延长EAE临床进程，并维持降低的临床评分和保护慢性EAE期间丧失的轴突完整性。这些研究将进一步表征Axl和Mertk在EAE期间的作用，使用活化抗体作为靶向促炎信号分子的可能治疗方法。这种更精细的方法将靶向两种TAM，因为Gas 6以Gas 6对其受体Axl> Tyro 3>Mertk的相对亲和力激活所有三种TAM。我们将不研究Tyro 3，因为没有证据表明Tyro 3在炎症、EAE或MS病变期间发生变化。这种更有针对性的方法可以提供神经保护，防止慢性MS CNS组织中发现的轴突损伤和损失，并且可能作为患有进展性MS的个体的治疗是有益的。