Development of a Novel Method for Inhibiting Atherosclerosis in Diabetes

描述(申请人提供)：拟议研究的长期目标是开发一种可以安全地用于糖尿病患者的单抗，以确定它是否可以抑制动脉粥样硬化。胰岛素样生长因子-L(IGF-I)参与动脉粥样硬化病变增殖期的启动，但抑制IGF-I受体活性可能导致毒性作用。IGF-I与Av？3整合素协同作用，刺激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靶向AV？3整合素是否干扰IGF-I刺激的动脉粥样硬化病变进展。针对AV|33整合素03亚基的半胱氨酸环(氨基酸177-183)的特异性单抗将被制备并进行测试，以确定它是否抑制IGF-I刺激的SMC在体外的生长和迁移，以及这种反应是否由于IGF-I受体连接的信号机制的破坏而导致刺激细胞生长。为了确定这种抗体在体内是否有效，将制备抗体的Fab片段，并将其注入正在形成动脉粥样硬化病变的猪的动脉中。对照抗体将被注入其中一个病变区域，以排除由于自身免疫而产生非特异性影响的可能性。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的克隆，并使用RT-PCR扩增对应于重链和轻链的cDNA。将分离、克隆这些cDNA并测定它们的序列。抗体结构将用于在II期设计人源化形式的抗体。这些研究将最终验证通过抑制血管壁上AV？33的配体占位来抑制IGF-I作用的假说，从而抑制动脉粥样硬化病变的进展。如果这些结果表明这种方法是可行的，那么这种单抗的人源化版本将被制备、纯化并进行毒性测试。长期目标将是给患有动脉粥样硬化的糖尿病患者注射这种抗体，这些患者有广泛传播的弥漫性疾病，不适合目前的治疗方法，如药物洗脱支架。由于动脉粥样硬化是导致80%糖尿病患者死亡的原因，而且目前还没有专门针对糖尿病患者动脉粥样硬化的治疗方法，因此迫切需要针对这种并发症的治疗方法。

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.