Development of a novel method for inhibiting diabetic retinopathy

描述（由申请方提供）：拟定研究的长期目标是开发一种抑制糖尿病视网膜病变进展的单克隆抗体。胰岛素样生长因子-I（IGF-I）与内皮功能障碍以及疾病的增殖期有关。然而，直接抑制IGF-I受体可能导致神经毒性。暴露于高血糖后，内皮细胞利用PI-3和MAP激酶途径激活内皮细胞通透性和血管生成的变化。然而，与暴露于正常葡萄糖的细胞不同，这些途径通过需要跨膜蛋白SHPS-1的酪氨酸磷酸化的替代机制响应IGF-I而被激活。SHPS-1被IGF-I受体直接磷酸化，导致信号蛋白复合物的募集。为了使这种复合物在高血糖加IGF-I的反应中被激活，它必须与称为整合素相关蛋白（IAP）的膜蛋白结合。我们的初步研究表明，在早期视网膜病变，毛细血管渗漏和白细胞停滞可以通过破坏IAP/SHPS-1的关联来抑制。另外，尽管存在长期糖尿病，IAP敲除小鼠不发展增加的毛细血管通透性，因此看来IAP/SHPS-1缔合是视网膜病变进展在体内所需的。这些研究的目的是确定破坏IAP/SHPS-1是否导致SHPS-1复合物在内皮细胞上正常组装失败，并激活介导内皮功能障碍的适当下游信号分子。我们将确定直接针对大鼠IAP中氨基酸71-80的多克隆抗体是否具有体内效力。将纯化的抗体注射到糖尿病大鼠体内，并测定其对毛细血管渗漏、白细胞停滞和内皮细胞凋亡的影响。随后，将开发并测试针对人IAP中氨基酸71-80的单克隆抗体，以确定其是否抑制IGF-I刺激的信号传导以及内皮管形成和细胞渗透性的变化。为了获得可以安全地施用于人的单克隆抗体，将需要使鼠形式人源化。将分离分泌鼠抗体的克隆，并使用rtPCR扩增对应于重链和轻链的cDNA，然后对cDNA进行测序。这些研究应该明确地验证这样的假设，即通过抑制培养的内皮细胞和大鼠毛细血管中的SHPS-1/IAP结合来抑制IGF-I的作用是糖尿病视网膜病变中发生的病理生理变化。如果这些研究的结果表明抗体有效，则在II期研究期间，将制备、纯化单克隆抗体的人源化形式，并测定其效果。长期目标是将这种抗体用于有早期视网膜病变迹象的糖尿病患者，以确定其是否 在抑制该疾病的早期事件和预防晚期症状方面具有功效。由于糖尿病视网膜病变是工作年龄成人失明的主要原因，因此需要针对该并发症的治疗的新疗法。 公共卫生相关性：本发明的目的在于开发单克隆抗体作为糖尿病性视网膜病变患者的给药药物。糖尿病视网膜病变是工作年龄成人（25-74岁）失明的主要原因。因此，它是一个重大的公共卫生问题。目前可用的抑制该问题的药物对约1/3的患者有效。待开发的药物通过与这些药物不同的机制发挥作用，因此有可能改善2/3受影响患者的预后。本提案中的工作将决定 如果这种方法是可行的，使用这种疾病状态的动物模型。

DESCRIPTION (provided by applicant): The long-term objective of the proposed study is to develop a monoclonal antibody that inhibits the progression of diabetic retinopathy. Insulin-like growth factor-I (IGF-I) has been implicated in both endothelial dysfunction as well as the proliferative phase of the disease. However directly inhibiting the IGF-I receptor may be lead to neurotoxicity. Following exposure to hyperglycemia, endothelial cells utilize both the PI-3 and MAP kinase pathways to activate changes in endothelial cell permeability and angiogenesis. However unlike cells exposed to normal glucose these pathways are activated in response to IGF-I by an alternative mechanism that requires tyrosine phosphorylation of the transmembrane protein, SHPS-1. SHPS-1 is phosphorylated directly by the IGF-I receptor leading to recruitment of a complex of signaling proteins. For this complex to be activated in response to hyperglycemia plus IGF-I, it must associate with a membrane protein termed integrin associated protein (IAP). Our preliminary studies have shown that in early retinopathy, capillary leakage and leukostasis can be inhibited by disrupting IAP/SHPS-1 association. Additionally IAP knockout mice do not develop increased capillary permeability in spite of longstanding diabetes therefore it appears that IAP/SHPS-1 association is required in vivo for retinopathy progression. The purpose of these studies will be to determine if disrupting IAP/SHPS-1 results in failure of the SHPS-1 complex to assemble normally on endothelial cells and activate the appropriate downstream signaling molecules that mediate endothelial dysfunction. We will determine if a polyclonal antibody direct against amino acids 71-80 in rat IAP has efficacy in vivo. The purified antibody will be injected into diabetic rats and its effects on capillary leakage, leukostasis and endothelial cell apoptosis determined. Subsequently a monoclonal antibody directed against amino acids 71-80 in human IAP will be developed and tested to determine if it inhibits IGF-I stimulated signaling as well as changes in endothelial tube formation and cell permeability. To obtain a monoclonal antibody that can be administered to humans safely it will be necessary to humanize the murine form. A clone secreting the murine antibody will be isolated and cDNAs corresponding to heavy and light chains will be amplified using rtPCR then the cDNAs will be sequenced. These studies should definitively test the hypothesis that inhibiting IGF-I actions by inhibiting SHPS-1/IAP association in endothelial cells in culture and in capillaries in rats inhibis pathophysiologic changes that occur in diabetic retinopathy. If the results of these studies show that the antibody is effective, during phase II studies a humanized form of the monoclonal antibody will be prepared, purified and its effects determined. The long term objective would be to administer this antibody to diabetic patients with early signs of retinopathy to determine if it has efficacy in inhibiting both the early events and preventing late stage manifestations of this disease. Since diabetic retinopathy is the leading cause of blindness in working age adults, there is a need for new therapies that are directed toward treatment of this complication. PUBLIC HEALTH RELEVANCE: This proposal is directed toward the development of a monoclonal antibody as a drug to be administered to patients with diabetic retinopathy. Diabetic retinopathy is the leading cause of blindness in working age adults (25-74 years). As such it represents a significant public health problem. Drugs that are currently available that inhibit thi problem are effective in approximately 1/3 of patients. The drug to be developed functions by a mechanism that is different than those drugs and therefore has the potential to improve the prognosis of this disease in 2/3 of the affected patients. The work in this proposal will determine if this approach is feasible using animal models of this disease state.