Targeted Enzymes for Prodrug Therapy

用于前药治疗的靶向酶

• 批准号: 7120096
• 负责人: DAVID N KRAG
• 金额: $ 54.73万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-07 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7120096
• 关键词: SCID mouse; antineoplastic antibiotics; apoptosis; beta lactamase; breast neoplasms; cell line; cephalosporins; clinical research; combination chemotherapy; cytotoxicity; doxorubicin; drug design /synthesis /production; drug screening /evaluation; enzyme linked immunosorbent assay; enzyme therapy; human tissue; immunoconjugates; neoplasm /cancer chemotherapy; neoplasm /cancer immunotherapy; nonhuman therapy evaluation; phage display; pharmacokinetics; polymerase chain reaction; prodrugs; protein engineering; surface plasmon resonance

DESCRIPTION (provided by applicant): Using phage-displayed random enzyme libraries, our goal is to develop tumor-targeted effector molecules that convert prodrugs to toxins for cancer therapy. The rationale for this approach is based on published experiments with single-chain antibody (scFv) targeted enzymes that specifically activate prodrugs of potent cytotoxic agents. These prodrugs have significantly less cytotoxicity than the toxins released by cleavage with the targeted enzyme. We hypothesize that phage enzymes with the targeting moiety built directly into the protein scaffold when panned against tumor cells isolated from patients following surgical resection will contain tumor selective effector molecules. The benefits of panning enzyme libraries on fresh human tumor cells include the presence of a vast number of possible targets, targets will be in their native configuration, subtraction of the pools of ligands binding to normal tissue, and the ability to rapidly test these novel targeting agents for efficacy using a growing portfolio of prodrugs. The Specific Aims are 1) Generate diverse randomized loop libraries of enzymes on phage 2) Surgically sample tumor tissue, isolate small numbers of fresh tumor cells, and identify phage enzymes that have bound selectively to tumor cells using a novel method of PCR-based phage display, 3) Determine whether candidate enzymes bind selectively to tumor tissue specimens and not to normal tissues using a novel fluorometric substrate, and 4) Characterize targeted enzyme activation of cytotoxic prodrugs in vitro and in vivo in animal models. Successful completion of these experiments will result in demonstration of novel targeted effector molecules that bind selectively to tumors and activate cytotoxic prodrugs.

描述（由申请人提供）：使用噬菌体展示的随机酶文库，我们的目标是开发肿瘤靶向效应分子，将前药转化为毒素以用于癌症治疗。这种方法的基本原理是基于已发表的单链抗体 (scFv) 靶向酶实验，这些酶特异性激活强效细胞毒性药物的前药。这些前药的细胞毒性明显低于目标酶裂解释放的毒素。我们假设，当针对手术切除后从患者分离的肿瘤细胞进行淘选时，直接构建到蛋白质支架中的具有靶向部分的噬菌体酶将含有肿瘤选择性效应分子。在新鲜人类肿瘤细胞上淘选酶库的好处包括存在大量可能的靶标，靶标将处于其天然构型，减去与正常组织结合的配体池，以及能够使用不断增长的前药组合快速测试这些新型靶向剂的功效。具体目标是 1) 在噬菌体上生成不同的随机循环酶文库 2) 通过手术对肿瘤组织进行取样，分离少量新鲜肿瘤细胞，并使用基于 PCR 的噬菌体展示的新方法鉴定与肿瘤细胞选择性结合的噬菌体酶，3) 使用新型荧光底物确定候选酶是否选择性结合肿瘤组织样本而不是正常组织，以及 4) 在动物模型中体外和体内表征细胞毒性前药的靶向酶激活。这些实验的成功完成将证明新型靶向效应分子可以选择性地结合肿瘤并激活细胞毒性前药。