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AFFINITY MATURATION OF IL-2 AND RELATED PEPTIDES

IL-2 和相关肽的亲和力成熟

基本信息

批准号：
2750022
负责人：
THOMAS L CIARDELLI
金额：
$ 19.34万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-08-01 至 2000-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2750022
关键词：
bacterial virus chemical synthesis cytokine receptors genetic library human subject interleukin 2 peptide analog protein structure function receptor binding

项目摘要

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，对免疫系统内蛋白质信使的研究而造血系统已经导致确认了超过25个具有调节功能的不同蛋白质分子。白介素2 (IL-2)是最早发现的免疫系统激素之一，一直是最深入的调查之一。此外，IL-2是临床上用于包括抗肿瘤在内的各种应用以及抗艾滋病毒试验。白介素2的构效关系 IL-2受体系统也一直是许多研究的重点。然而，目前还没有有效的IL-2拮抗剂或超级激动剂据报道。这部分是由于IL-2受体的复杂性。系统，其第三个亚单位最近才被识别，并且事实上，IL-2的一个长期存在的3埃X射线结构最近被证明是严重错误的。尽管有这些限制，我们的实验室进行了结构功能研究，结果是用部分拮抗剂和超激动剂制备IL-2类似物属性。然而，与进步相比，这些影响是微弱的。在相关细胞因子人类生长激素的合成孔径雷达研究中取得进展采用了哪种更强大的方法，如噬菌体展示。因此，我们集中了最近的努力开发所需的工具，使我们能够开始一种新的方法来用同样的噬菌体展示技术制备高效的1L-2类似物方法论。我们成功地表达了这三个受体亚基胞外域作为选择介质。我们开发了一种全新的可溶受体复合体的设计和制备方法探讨将IL-2与细胞表面型亲和力结合。这些复合体可能被证明是随机选库在图书馆中成功应用的关键 IL-2受体系统。我们已经从我们的之前的研究将被用来代替野生型IL-2，每项研究模板具有独特的活动，应有助于准备更多强有力的类比。最后，我们开发了一种多肽模板，它将假定溶液中的构象与噬菌体表面的构象相同。此模板上下文中的随机库应该会导致选择较小多肽形式的IL-2拮抗剂。我们的具体目标是： A.利用“噬菌体展示”方法的多功能性受体结合增强的IL-2类似物的制备属性作为通向激动剂和拮抗剂的途径。 B.使用稳定的螺旋发夹多肽序列作为模板可维护的随机表面拓扑库的噬菌体展示在溶液中显示受体结合构象。