Development of a Novel Method for Inhibiting Atherosclerosis in Diabetes

描述（由申请方提供）：拟定研究的长期目标是开发一种可安全用于糖尿病患者的单克隆抗体，以确定其是否可抑制动脉粥样硬化。胰岛素样生长因子-I（IGF-I）已涉及启动动脉粥样硬化病变发展的增殖期，但抑制IGF-I受体活性可能导致毒性。IGF-I的功能与AV合作？3整合素刺激平滑肌细胞（SMC）分裂和迁移，与动脉粥样硬化的发生发展有关。因此，这些研究的目的将是确定是否针对AV？3整合素干扰IGF-I刺激的动脉粥样硬化病变进展。针对aV 03亚基半胱氨酸环（氨基酸177-183）的特异性单克隆抗体|将制备并测试33整联蛋白，以确定其是否在体外抑制IGF-I刺激的SMC生长和迁移，以及该反应是否是由于导致刺激细胞生长的IGF-I受体相关信号传导机制的破坏。为了确定该抗体是否具有体内功效，将制备抗体的Fab片段并将其输注到正在发展动脉粥样硬化病变的猪的动脉中。将对照抗体输注到其中一个病变区域，以排除自身免疫引起非特异性效应的可能性。为了获得制备人源化抗体所需的结构信息，将分离分泌抗体的细胞克隆，并使用RTPCR扩增与重链和轻链对应的cDNA。将分离、克隆cDNA并测定其序列。该抗体结构将用于设计II期抗体的人源化形式。这些研究应明确测试的假设，抑制IGF-I的行动，抑制配体占用的AV？33抑制动脉粥样硬化病变进展。如果这些结果表明该方法是可行的，则将制备、纯化该单克隆的人源化版本并测试毒性。长期目标是将这种抗体给予患有动脉粥样硬化的糖尿病患者，这些患者患有广泛的弥漫性疾病，这些疾病不适合目前的治疗，如药物洗脱支架。由于动脉粥样硬化是80%的糖尿病患者的死因，并且目前还没有专门治疗糖尿病患者动脉粥样硬化的有效疗法，因此非常需要针对这种并发症的治疗的新疗法。

DESCRIPTION (provided by applicant): The long term objective of the proposed studies is to develop a monoclonal antibody that can be administered safely to patients with diabetes to determine if it can inhibit atherosclerosis. Insulin-like growth factor-l (IGF-I) has been implicated in initiating the proliferative phase of atherosclerotic lesion development but inhibiting IGF-I receptor activity may lead to toxicity. IGF-I functions cooperatively with the aV?3 integrin to stimulate smooth muscle celll(SMC) division and migration which are related to the development of atherosclerosis. Therefore the purpose of these studies will be to determine if targeting the aV?3 integrin disrupts IGF-I stimulated atherosclerotic lesion progression. A specific monoclonal antibody directed against the cysteine loop (amino acids 177-183) of the 03 subunit of the aV|33 integrin will be prepared and tested to determine if it inhibits IGF-I stimulated SMC growth and migration in vitro and if this response is due disruption of IGF-I receptor linked signaling mechanisms that lead to stimulation of cell growth. To determine if this antibody has efficacy in vivo Fab fragments of the antibody will be prepared and will be infused into the arteries of pigs that are developing atherosclerotic lesions. A control antibody will be infused into one of the lesion areas to rule out the possibility of nonspecific effects due to autoimmunization. To obtain the structural information that will be necessary to prepare a humanized form of the antibody the clone of cells secreting the antibody will be isolated and the cDNAs corresponding to the heavy and light chains will be amplified using RTPCR. The cDNAs will be isolated, cloned and their sequences determined. The antibody structure will be used to design humanized form of the antibody in phase II. These studies should definitively test the hypothesis that inhibiting IGF-I actions by inhibiting ligand occupancy of aV?33 in blood vessel walls inhibits atherosclerotic lesion progression. If these results show that this approach is feasible a humanized version of this monoclonal would be prepared, purified and tested for toxicity. The long term objective would be to administer this antibody to diabetic patients with atherosclerosis who have wide spread diffuse disease that is not amenable to current therapies such as drug eluting stents. Since atherosclerosis is the cause of death in 80% of diabetics and there is no currently available therapy that specifically treats atherosclerosis in diabetics there is a major need for new therapies that are directed toward the treatment of this complication.