BIOSPECIFIC POLYMER ENZYME CONJUGATES FOR DRUG DELIVERY

用于药物输送的生物特异性聚合物酶缀合物

• 批准号: 6489389
• 负责人: Glen S. Kwon
• 金额: $ 9.82万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-08 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6489389
• 关键词: SDS polyacrylamide gel electrophoresis; affinity chromatography; amination; antitumor antibody; binding proteins; biotin; carboxypeptidase; chemical conjugate; drug delivery systems; drug screening /evaluation; enzyme activity; enzyme linked immunosorbent assay; immunoglobulin G; laboratory mouse; monoclonal antibody; neoplasm /cancer immunotherapy; nonhuman therapy evaluation; polyethylene glycols; polymers; squamous cell carcinoma

Description: (Applicant's abstract) Once a target location has been identified for a drug, protein or gene, proper spatial and temporal control in the delivery of these molecules is a fundamental problem in biomedical engineering. In one promising approach for anticancer drugs called antibody-directed enzyme prodrug therapy (ADEPT), a monoclonal antibody (MAb)-enzyme conjugate selectively binds an antigen expressed on tumor cells, and the enzyme moiety releases drug from the subsequently injected prodrug at the target site. ADEPT is in clinical trials. However, drawbacks of MAb-enzyme conjugates limit ADEPT. They express low chemical and physical stability, short blood-half life, immunogenicity and low tumor to blood ratio. Attaching a common water-soluble polymer, methoxy-terminated poly(ethylene glycol) (PEG), onto MAb-enzyme conjugates enhances stability, prolongs blood circulation and reduces immunogenicity, but with no marked increase in tumor to blood ratio. Our research will focus on biospecific polymer-enzyme conjugates and their role in ADEPT. We attached a biotinylated PEG on a model enzyme, carboxypeptidase A (CPA). A biotin moiety at a chain end of PEG may mediate several useful functions for the first time for a PEG-enzyme conjugate. A biotin moiety may mediate the separation of PEG-CPA conjugate by affinity chromatography, fractionating in terms of number of attached biotinylated PEG on CPA using an immobilized monomeric avidin. A biotin moiety may tether an antibody in conjunction with biotin and streptavidin. Lastly, a biotin moiety may bind a clearing agent (e.g., streptavidin) in blood, an interaction that may mediate the clearance of biotinylated PEG-CPA conjugate by the liver and an increase in its tumor to blood ratio. The specific aims of the proposal: (1) To prepare a biotinylated PEG-CPA conjugate with controlled levels of biotinylated PEG at varied molecular weight by reductive amination and by affinity chromatography with immobilized monomeric avidin. (2) To study the catalytic activity and the stability of fractionated biotinylated PEG-CPA conjugates. (3) To study the immunogenicity and the plasma profile of fractionated biotinylated PEG-CPA conjugates in mice, focusing on their clearance by streptavidin. (4) To tether an IgG1 (174H.64) together with biotin and streptavidin on biotinylated PEG-CPA conjugate with optimized properties, purify the conjugate to obtain a 1:1 complex, study its stability, and assess target cell binding in vitro. (5) To study the plasma profile and the biodistribution of antibody-biotinylated PEG-CPA conjugates ("active" targeting) and biotinylated PEG-CPA conjugates ("passive" targeting) in tumor-bearing mice (KLN-205), assessing the effect of injected streptavidin.

描述：（申请人的摘要）一旦确定了目标位置 对于药物，蛋白质或基因，适当的空间和时间控制 这些分子的传递是生物医学工程中的一个基本问题。 在一种有前途的抗癌药物中，称为抗体定向酶 前药治疗（Adept），一种单克隆抗体（MAB） - 酶结合物 有选择地结合在肿瘤细胞上表达的抗原，酶部分 从随后在目标部位注射的前药中释放药物。熟练 正在临床试验中。但是，mab-酶的偶联物的缺点限制了熟练。 它们表达低化学和身体稳定性，血半寿命短， 免疫原性和低肿瘤与血比。附着一个普通的水溶性 聚合物，甲氧基终止的聚乙二醇（PEG），上 共轭增强稳定性，延长血液循环并减少 免疫原性，但肿瘤与血比没有明显增加。 我们的研究将重点放在生物特异性聚合物 - 酶结合物及其作用上 熟练。我们在模型酶，羧肽酶A上附着一个生物素化的钉 （CPA）。 PEG链端处的生物素部分可能会介导一些有用的 首次进行钉 - 酶结合物的功能。生物素部分可能 通过亲和力色谱法介导PEG-CPA结合物的分离， 使用A A A上附着的生物素化PEG的数量来分馏 固定的单体抗原。生物素部分可能会在 与生物素和链霉亲和素结合。最后，生物素部分可以结合 血液中的清除剂（例如链霉亲和素），这种相互作用可能介导 肝脏的生物素化PEG-CPA结合的清除和增加 它的肿瘤与血比。提案的具体目的：（1）准备一个 生物素化的PEG-CPA结合物与受控水平的生物素化钉在 通过还原胺和亲和色谱分子量变化的分子量变化 与固定的单体抗原蛋白。 （2）研究催化活性和 分离生物素化的PEG-CPA结合物的稳定性。 （3）研究 免疫原性和分离生物素化PEG-CPA的血浆谱 偶联的小鼠，专注于链霉生蛋白酶的清除。 （4）系绳 IgG1（174h.64）与生物素和链霉亲素一起生物素化PEG-CPA 与优化特性共轭，净化共轭以获得1：1 复杂，研究其稳定性并评估体外的靶细胞结合。 （5）到 研究血浆谱和生物分布抗体 - 生物素化 PEG-CPA结合物（“主动”靶向）和生物素化的PEG-CPA结合物 （“被动”靶向）肿瘤小鼠（KLN-205），评估 注射链霉亲和素。