AFFINITY MATURATION OF IL-2 AND RELATED PEPTIDES

IL-2 和相关肽的亲和力成熟

• 批准号: 2192042
• 负责人: THOMAS L CIARDELLI
• 金额: $ 20.19万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2192042
• 关键词: bacterial virus; chemical synthesis; cytokine receptors; genetic library; human subject; interleukin 2; peptide analog; protein structure function; receptor binding

Over the past 15 years, the study of protein messengers within the immune and hematopoietic systems has lead to the identification of more than 25 distinct protein molecules possessing regulatory function. Interleukin -2 (IL-2) was one of the first immune system hormones to be discovered and has been one of the most intensely investigated. In addition, IL-2 is being used clinically for a variety of applications including anti - tumor and anti - HIV trials. Structure - activity relationships for the IL-2 - IL-2 receptor system have also been the focus of many studies. Nevertheless, no potent IL-2 antagonists or superagonists have been reported. This has been in part due to the complexity of the IL-2 receptor system, the third subunit of which was only recently identified, and the fact a long standing 3 Angstroms X-ray structure for IL-2 was also recently shown to be seriously in error. Despite these limitations, our laboratory has pursued structure function studies that have resulted in the preparation of IL-2 analogs with partial antagonist and superagonist properties. These effects are weak, however, when compared to the progress achieved in SAR studies of the related cytokine, Human Growth Hormone, in which more powerful methods such as phage display have been employed. Consequently, we have concentrated our recent efforts developing the required tools that will allow us to begin a new approach to the preparation of potent 1L-2 analogs using the same phage display methodology. We have successfully expressed all three receptor subunit ectodomains to serve as selection media. We have developed an entirely new approach to the design and preparation of soluble receptor complexes which bind IL-2 with cell surface-like affinity. These complexes may prove to be the key to the successful application of random library selection in the IL-2 receptor system. We have identified several templates from our previous studies that will be employed instead of wildtype IL-2, each template has a unique activity that should facilitate preparation of more potent analogs. Finally, we have developed a peptide template that will assume the same conformation in solution as on the surface of phage. Random libraries in the context of this template should lead to the selection of IL-2 antagonists in a smaller peptide format. Our Specific Aims are: A. To exploit the versatility of "phage display" methodology for the preparation of IL-2 analogs with greatly enhanced receptor binding properties as a route to both agonists and antagonists. B. To employ a stable helical hairpin peptide sequence as a template for phage display of random surface topology libraries capable of maintaining the displayed receptor binding conformation in solution.

在过去的15年里，免疫系统中蛋白质信使的研究 和造血系统已经导致超过25个 具有调节功能的不同蛋白质分子。白细胞介素-2 （IL-2）是最早被发现的免疫系统激素之一， 一直是研究最深入的领域之一此外，IL-2是 临床上用于多种应用，包括抗肿瘤 和抗艾滋病毒试验。IL-2的结构-活性关系 IL-2受体系统也一直是研究的热点。 然而，还没有有效的IL-2拮抗剂或超激动剂被用于治疗IL-2。 报道这部分是由于IL-2受体的复杂性 系统，其中第三个亚基是最近才确定的， 事实上，IL-2的长期存在的3 Angiography X射线结构也是 最近被证明是严重错误的。尽管有这些限制，我们 实验室进行了结构功能研究， IL-2部分拮抗剂和超激动剂类似物制备 特性.然而，与进步相比，这些影响是微弱的。 在相关细胞因子，人生长激素， 已经采用了哪些更有效的方法如噬菌体展示。 因此，我们最近集中精力发展 所需的工具，使我们能够开始一个新的方法， 使用相同噬菌体展示制备有效的1 L-2类似物 方法论我们已经成功表达了所有三种受体亚基， 胞外域作为选择介质。我们开发了一种全新的 设计和制备可溶性受体复合物的方法， 以细胞表面样亲和力结合IL-2。这些复合物可能被证明是 随机选库成功应用的关键是 IL-2受体系统。我们已经确定了几个模板， 以前的研究将采用替代野生型IL-2， 模板具有独特活动， 有效的类似物。最后，我们开发了一种肽模板， 在溶液中呈现与在噬菌体表面上相同的构象。 这个模板上下文中的随机库应该导致 选择较小肽形式的IL-2拮抗剂。 我们的具体目标是： A.为了利用“噬菌体展示”方法的多功能性， 受体结合大大增强的IL-2类似物的制备 作为激动剂和拮抗剂的途径的性质。 B。采用稳定的螺旋发夹肽序列作为模板， 噬菌体展示随机表面拓扑文库 在溶液中显示的受体结合构象。