BIOSPECIFIC POLYMER ENZYME CONJUGATES FOR DRUG DELIVERY

描述：(申请人摘要)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 这些分子的输送是生物医学工程中的一个基本问题。 在一种名为抗体导向酶的抗癌药物开发方法中 前药物治疗(ADEPT)，一种单抗(MAb)-酶结合物 选择性地结合在肿瘤细胞上表达的抗原，以及酶部分 在靶部位从随后注射的前药中释放药物。熟练的 正在进行临床试验。然而，单抗-酶结合物的缺点限制了它的应用。 它们表现出化学和物理稳定性低，血液半衰期短， 免疫原性和低瘤血比。附着一种常见的水溶性 端甲氧基聚乙二醇单抗酶聚合物 结合物增强稳定性，延长血液循环，减少 免疫原性，但肿瘤与血液的比率没有明显增加。 我们的研究将集中在生物特异性聚合物-酶偶联物及其作用 熟练地。我们将生物素化的聚乙二醇化的聚乙二醇固定在模型酶--羧基肽酶A上 (注册会计师)。聚乙二醇链末端的生物素部分可能介导几种有用的 第一次为聚乙二醇酶偶联物起作用。生物素部分可以 以亲和层析为中介分离聚乙二醇偶联物， 以生物素化聚乙二醇乙二醇酯在CPA上的附着数量为单位进行分级 固定化单体亲和素。生物素部分可以将抗体锚定在 与生物素和链霉亲和素结合使用。最后，生物素部分可以结合一个 血液中的清除剂(例如链霉亲和素)，一种可能调节 生物素标记的聚乙二醇CPA偶联物在肝脏中的清除和增加 它的肿瘤与血液的比率。该提案的具体目标是：(1)编制一份 生物素化的聚乙二醇-CPA偶联物与生物素化的聚乙二醇在 通过还原胺化和亲和层析改变分子量 用固定化单体亲和素。(2)研究了催化剂的催化活性和催化性能。 分级生物素化聚乙二醇CPA偶联物的稳定性。(3)研究 生物素化聚乙二醇单抗的免疫原性及血浆谱 小鼠体内的结合物，重点是链霉亲和素对它们的清除。(4)系绳 结合生物素和链霉亲和素的免疫球蛋白1(174H.64)在生物素标记的聚乙二醇单抗上的应用 优化性能的偶联物，纯化得到1：1的偶联物 研究其稳定性，并在体外评估靶细胞结合情况。(5)至 抗体生物素标记物的血浆谱和生物分布研究 聚乙二醇-环丙交联物(“活性”靶向)和生物素化的聚乙二醇单环-环丙交联物 (“被动”靶向)在荷瘤小鼠(KLN-205)中，评估 注射链霉亲和素。

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.