Glycine rich sequences with pharmacokinetic enhancing properties of PEG polymers

描述（由申请人提供）：蛋白质药物已被批准用于许多治疗适应症，代表了制药行业快速增长的部分。然而，由于次优的药代动力学特性和免疫原性问题，许多批准的蛋白质药物和开发中的候选物未能达到其潜在功效。这些特性包括短循环半衰期、短保存期、低溶解度、快速肾清除和对蛋白水解降解的敏感性。用亲水性化学聚合物如聚乙二醇（PEG）修饰蛋白质是一种经临床验证的解决这些限制的方法。然而，与连接这些聚合物所需的化学改性程序相关的挑战提出了重大挑战。我们的最终目标是产生模拟亲水性化学聚合物如PEG的理化性质的氨基酸序列。我们的建议是基于这样的观察，即富含甘氨酸的序列（GRS），其中含有很少的疏水性氨基酸将不会折叠成紧凑的三维结构，但会采取随机构象与大的流体动力学半径类似的PEG。我们假设，当它们与治疗性蛋白质连接时，将赋予类似的药代动力学改善。这些序列可以使用常规重组技术连接到蛋白质上，因此完全避免了化学修饰步骤的需要。我们已经合成了一个198个氨基酸的甘氨酸丰富的序列的基础上出现在人类蛋白质的序列。我们的目标是表达这种蛋白质，并系统地测试其与药代动力学增强相关的理化和生物学特性。我们的具体目标是：1）在E.大肠杆菌表达系统进行下游表征和研究。2)表征GRS蛋白的血清稳定性、蛋白酶抗性和生物物理特性。3)在动物模型中表征GRS蛋白的血浆药代动力学和免疫原性潜力。在II期，我们将对GRS进行详细优化，以实现高水平表达、血浆半衰期延长和免疫原性降低。我们的目标是将优化的GRS融合到药物活性蛋白质如干扰素-α或G-CSF中进行动物和临床研究。我们的最终目标是验证并使这种方法易于应用于治疗性蛋白质。我们的项目旨在产生富含甘氨酸的序列，这些序列可以重组连接到治疗性蛋白质上，以改善其药代动力学特性。这种方法将避免与用亲水聚合物如PEG修饰蛋白质相关的困难。

DESCRIPTION (provided by applicant): Protein drugs have been approved for many therapeutic indications and represent a rapidly growing segment of the pharmaceutical industry. However, many approved protein drugs and candidates in development fail to reach their potential efficacy due to suboptimal pharmacokinetic properties and immunogenicity concerns. These properties include short circulating half-lives, short shelf lives, low solubility, rapid kidney clearance and susceptibility to proteolytic degradation. The modification of proteins with hydrophilic chemical polymers like polyethylene glycol (PEG) is a clinically validated approach to addressing these limitations. However, the challenges associated with chemical modification procedures required for the attachment of these polymers present significant challenges. Our ultimate goal is to generate amino acid sequences that mimic the physiochemical properties of hydrophilic chemical polymers like PEG. Our proposal is based on the observation that glycine rich sequences (GRS) which contain few hydrophobic amino acids will not fold into compact 3-dimensional structures but will adopt random conformations with large hydrodynamic radii similar to PEG. We hypothesize that they will confer similar pharmacokinetic improvements when attached to therapeutic proteins. These sequences can be attached to proteins using conventional recombinant technology and thus completely obviates the need for chemical modifications steps. We have synthesized a 198 amino acid glycine rich sequence based on sequences that occur in human proteins. We aim to express this protein and systematically test its physiochemical and biological properties relevant to pharmacokinetic enhancement. Our specific aims are: 1) Produce 5 mg of a purified GRS protein in E. coli expression system for downstream characterization and studies. 2) Characterize serum stability, protease resistance and biophysical properties of the GRS protein. 3) Characterize plasma pharmacokinetics and immunogenic potential of the GRS protein in animal models. In Phase II, we will perform detailed optimization of GRS for high-level expression, plasma half-life extension and reduced immunogenicity. We aim to advance optimized GRS which are fused to pharmaceutically active proteins like interferon-alpha or G-CSF into animal and clinical studies. Our ultimate goal is to validate and make this method readily applicable to therapeutic proteins. Our project aims to generate glycine rich sequences that can be attached recombinantly to therapeutic proteins to improve their pharmacokinetic properties. This approach would circumvent the difficulties associated with the modifying proteins with hydrophilic polymers like PEG.