Zwitterionic Polypeptide-Protein Conjugation for the Safe and Efficient Delivery of Therapeutic Enzymes

两性离子多肽-蛋白质缀合 安全有效地输送治疗酶 项目摘要 阻碍治疗性蛋白质产品广泛应用的主要障碍之一是它们的潜力 免疫应答，特别是从非人类来源获得的那些。目前，最 减轻外源蛋白诱导的免疫应答的成功策略是“PEG化”，即，以屏蔽 用聚乙二醇（PEG）修饰蛋白表面表位。这种表面缀合策略已经被 显示在一定程度上降低对潜在蛋白质的免疫应答，并且超过10种PEG化的 蛋白质产品已被食品和药物管理局（FDA）批准。但最近的研究 在动物模型和临床试验中均证明存在诱导的抗PEG抗体 在重复给药和预先存在的抗PEG抗体后， 这种聚乙二醇化技术。我们认为目前的聚乙二醇化存在两个缺点 技术：1）导致抗PEG抗体的两亲性PEG的半抗原特性，和2）非- 两亲性PEG的降解特性导致肾脏和肝脏空泡化。此前，我们开发了 一种两性离子聚（羧基甜菜碱）（PCB）保护的尿酸酶，它被证明能够维持 蛋白质生物活性，降低包裹蛋白质的免疫原性并延长其循环时间 没有诱导的抗聚合物抗体。从两性离子概念延伸，聚（EK）（PEK），一种两性离子 由选择性赖氨酸（K）和谷氨酸（E）组成的多肽，具有两性离子PCB的所有性质 并且也是可降解的以促进其从体内消除，而PEG或PCB不是。因此，PEK是一种 聚乙二醇化是一个伟大的替代品，但从未被研究过其在药物递送中的应用。的 该R21提案的目的是探索可降解的两性离子聚（EK）（PEK）-蛋白质的潜力 共轭作为PCB的可降解版本，预期PEK将提供与PCB类似的益处， 改性蛋白质药物，同时具有良好的生物降解性。我们的初步结果表明，PEK 即使将其偶联到高免疫原性匙孔帽贝上， 血蓝蛋白（KLH）通常用于增强半抗原的免疫原性。然而，显著的抗PEG 在平行研究中，通过PEG缀合的KLH引发抗体。在这项提案中，尿酸酶，一种非人类的 具有强免疫原性的酶，将被用作蛋白质靶标。我们只是假设， 这项研究表明，PEK-尿酸酶缀合物将提供一种安全有效的酶递送策略， 降低免疫原性、改善药代动力学（PK）和增强药效学（PD）。以来 聚乙二醇化修饰已广泛应用于蛋白质治疗学，本项目的成功完成将使聚乙二醇化修饰成为蛋白质治疗学的一个新的发展方向。 在一种新的方法中，作为聚乙二醇化技术的替代或替代， 和更有效的酶疗法。

Zwitterionic Polypeptide-protein Conjugation for the Safe and Efficient Delivery of Therapeutic Enzymes PROJECT SUMMARY One of the major obstacles that impede the wide application of therapeutic protein products is their potential immunological response, especially for those obtained from non-human sources. Currently, the most successful strategy to mitigate immune response induced by foreign proteins is “PEGylation”, i.e., to shield the protein surface epitopes with polyethylene glycol (PEG). This surface conjugation strategy has been shown to decrease to some extent immune responses to the underlying protein and more than ten PEGylated protein products have been approved by the Food and Drug Administration (FDA). However, recent studies both in animal models and clinical trials have demonstrated the presence of induced anti-PEG antibodies after repeated administrations and pre-existing anti-PEG antibodies, which directly challenge the future of this PEGylation technology. We believe that there are two shortcomings for the current PEGylation technology: 1) the haptenic character of amphiphilic PEG leading to anti-PEG antibodies and 2) non- degradable character of amphiphilic PEG leading to vacuolation of kidney and liver. Previously, we developed a zwitterionic poly(carboxybetaine) (PCB) protected uricase, which was shown to be capable of maintaining protein bioactivity, reducing the immunogenicity of encased proteins and extending their circulation time without induced anti-polymer Abs. Extended from the zwitterionic concept, poly(EK) (PEK), a zwitterionic polypeptide comprising of alternative lysine (K) and glutamic acid (E), has all properties of zwitterionic PCB and is also degradable to promote its elimination from the body while PEG or PCB is not. Thus, PEK is a great alternative beyond PEGylation, but has never been studied for its application to drug delivery. The purpose of this R21 proposal is to explore the potential of degradable zwitterionic poly(EK) (PEK)-protein conjugates. As a degradable version of PCB, PEK is anticipated to provide similar benefits as PCB to its modified protein drugs while possessing excellent biodegradability. Our preliminary results showed that PEK did not induce any anti-PEK antibodies even when it was conjugated onto highly-immunogenic keyhole limpet hemocyanin (KLH) commonly used to boost the immunogenicity of haptens. However, significant anti-PEG antibodies were elicited by PEG-conjugated KLH in the parallel study. In this proposal, uricase, a non-human enzyme with strong immunogenicity, will be used as a protein target. We hypothesize and will confirm though this study that PEK-uricase conjugates will provide a safe and efficient strategy for enzyme delivery by greatly reducing immunogenicity, improving pharmacokinetics (PK), and enhancing pharmacodynamics (PD). Since PEGylation has been widely used in protein therapeutics, successful completion of this project will culminate in a new approach as an alternative to or a replacement over the PEGylation technology, producing safer and more effective enzyme therapeutics.