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， 这些分子的递送是生物医学工程中的基本问题。 在一种很有前途的抗癌药物的方法中， 前药疗法（ADEPT），单克隆抗体（MAb）-酶结合物 选择性结合肿瘤细胞上表达的抗原，并且所述酶部分 在靶位点从随后注射的前药释放药物。ADEPT 正在进行临床试验然而，MAb-酶缀合物的缺点限制了ADEPT。 它们表现出低的化学和物理稳定性，短的血液半衰期， 免疫原性和低肿瘤与血液比率。附着一种常见的水溶性 聚合物，甲氧基封端的聚（乙二醇）（PEG），到单克隆抗体-酶 结合物增强稳定性，促进血液循环， 免疫原性，但没有显着增加肿瘤与血液的比例。 我们的研究将集中在生物特异性聚合物-酶结合物及其作用 在ADEPT。我们将生物素化的PEG连接到模型酶羧肽酶A上 （CPA）。PEG链端的生物素部分可以介导几种有用的生物素结合。 首次用于PEG-酶缀合物。生物素部分可以 通过亲和层析介导PEG-CPA缀合物的分离， 根据CPA上连接的生物素化PEG的数量，使用 固定化单体亲和素。生物素部分可以将抗体拴系在生物素中。 与生物素和链霉亲和素结合。最后，生物素部分可以结合生物素。 清算代理（例如，链霉抗生物素蛋白），这种相互作用可能介导 肝脏对生物素化PEG-CPA偶联物的清除率以及 肿瘤与血液的比例建议的具体目标是：（1）编写一份 生物素化的PEG-CPA缀合物与生物素化的PEG的受控水平， 通过还原胺化和亲和色谱法改变分子量 用固定化的单体抗生物素蛋白。(2)研究了该催化剂的催化活性和 分级生物素化PEG-CPA缀合物的稳定性。(3)研究 分级生物素化PEG-CPA的免疫原性和血浆特征 在小鼠中的缀合物，重点是它们的清除链霉亲和素。(4)转换tether 生物素化PEG-CPA上的IgG 1（174H.64）与生物素和链霉亲和素 结合物，纯化结合物以获得1：1的 复合物，研究其稳定性，并评估体外靶细胞结合。(5)到 研究生物素化抗体的血浆分布和生物分布 PEG-CPA缀合物（“主动”靶向）和生物素化的PEG-CPA缀合物 在荷瘤小鼠（KLN-205）中进行（“被动”靶向），评估 注射了链霉亲和素

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.