Glycopeptide Fusions to Increase Serum Half-life of Therapeutic Proteins

描述(由申请人提供)：项目摘要/摘要：注射的治疗性蛋白质的疗效往往因其快速从循环中清除而受到影响。这意味着必须更频繁地给予剂量，影响患者的依从性和治疗成本。学术和工业实验室正在进行一项重大研究，以发现如何修改蛋白质药物，使其在体内的停留时间延长。方法包括对蛋白质治疗药物进行化学修饰，或者创造含有额外氨基酸的蛋白质药物，或者实际上是整个蛋白质的药物，旨在增加半衰期。本建议的长期目标是提供一种原则上适用于所有治疗性蛋白质的方法，以延长它们在体内的停留时间，从而使它们更容易给药，并降低患者的成本。在这项应用中，我们建议探索引入肽序列，序列标签将允许在哺乳动物细胞系中重组生产过程中治疗蛋白的有限糖基化。假说是，这种增强糖基化的载体将保护蛋白质药物不被快速消除。我们的新序列标签来自人类蛋白质中已知发生糖基化的区域，这些区域应该允许碳水化合物掺入以延长蛋白质药物在体内的寿命。该提案的具体目标是：1.克隆并在HEK-293细胞中表达一种研究得很好的治疗蛋白的N-端和C-端延伸变体，其中延伸包括长度不同且已知经过糖基化的人类蛋白质序列；2.纯化蛋白质，并验证它们包含与加合物长度相对应的碳水化合物水平；3.证明N-和/或C-末端延伸没有取消生物活性；以及4.在动物模型中测试这些标记的蛋白质，以确定它们是否相对于天然母体蛋白药物增加了血清半衰期。该研究计划将针对这些具体目标而设计，并将把在HEK-293细胞中生产和表达新基因结构的专业知识与分子和细胞生物学方法结合起来，以及在蛋白质纯化和表征方面的专业知识，以生产可用于测试的可存活蛋白质。 公共卫生相关性：项目叙述：许多未解决和未得到满足的医疗需求的复杂性，如癌症、阿尔茨海默病、肌萎缩侧索硬化症和其他神经退行性疾病，增加了对蛋白质作为治疗这些疾病的药物的开发的关注。蛋白质药物由于制造成本高而昂贵，而且它们往往会迅速从体内消除，从而增加了剂量方案并增加了治疗费用。本提案提供了一种原则上适用于任何蛋白质药物的新方法，用于生产具有更长的活性持续时间(更少的剂量)和更低的成本的变体。

DESCRIPTION (provided by applicant): Project Summary/Abstract: The efficacy of injected therapeutic proteins is often compromised by their rapid clearance from the circulation. This means that doses must be administered more often, impacting both patient compliance and cost of treatment. A major search is underway in academic and industrial laboratories to discover ways to modify protein drugs in such a way that their residence time in the body is enhanced. Approaches include chemical modification of the protein therapeutic, or creation of protein drugs that contain stretches of additional amino acids, or indeed, of whole proteins, designed to promote increased half-life. The long-term objective of the present proposal is to provide a means that is applicable in principle to all therapeutic proteins for extending their residence time in the body, thus making them easier to administer and lower in cost to the patient. In this application we propose to explore the introduction of peptide sequences, sequence tags that will allow for limited glycosylation of the protein therapeutic during its recombinant production in a mammalian cell line. The hypothesis is that this vehicle for enhancing glycosylation will protect the protein drug from rapid elimination. Our novel sequence tags are derived from regions in a human protein that are known to undergo glycosylation and that should allow for incorporation of carbohydrate to enhance the lifetime of the protein drug in the body. The specific aims of the proposal are 1. To clone and express in HEK-293 cells, N- and C-terminally extended variants of a well-studied therapeutic protein in which the extensions comprise human protein sequences, varying in length and known to undergo glycosylation; 2. To purify the proteins to homogeneity and verify that they contain levels of carbohydrate that correspond to the length of the adduct; 3. To show that the N- and/or C-terminal extensions have not abolished biological activity; and 4. To test these tagged proteins in animal models to see if they increase serum half-life relative to the native parent protein drug. The research plan will be designed to address these specific aims and will combine expertise with methods of molecular and cell biology for production and expression of the new gene constructs in HEK-293 cells, together with expertise in protein purification and characterization to produce viable proteins for testing. PUBLIC HEALTH RELEVANCE: Project Narrative :The complexities of many unsolved and unmet medical needs such as cancer, Alzheimer's disease, ALS, and other neurodegenerative disorders has increased focus on development of proteins as drugs to treat these maladies. Protein drugs are expensive due to high cost of manufacture, and they tend to be rapidly eliminated from the body, thus increasing the dose regimen and adding to the price of treatment. The present proposal provides a novel method, applicable in principle to any protein drug, for producing variants with increased duration of activity (fewer doses), and reduced cost.