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）：使用噬菌体播放的随机酶库，我们的目标是开发针对肿瘤的效应子分子，这些分子将前药转化为毒素进行癌症治疗。这种方法的基本原理基于具有单链抗体（SCFV）靶向酶的已发表实验，该酶专门激活了有效的细胞毒性药物的前药。这些前药的细胞毒性明显少于与靶向酶裂解释放的毒素。我们假设，当对手术切除后从患者中分离的肿瘤细胞插入靶向部分时，靶向部分直接建立在蛋白质支架中的噬菌体将包含肿瘤选择性效应分子。平移酶库在新鲜的人类肿瘤细胞上的好处包括存在大量可能的靶标，目标将是其天然构型，与正常组织结合的配体池的减法以及快速测试这些新型目标的能力使用不断增长的前药组合的疗效代理。具体目的是1）在噬菌体上产生多种酶的随机循环库2）手术样品采样肿瘤组织，分离少量的新鲜肿瘤细胞，并鉴定使用基于PCR的新方法选择性地与肿瘤细胞结合的噬菌体酶显示，3）确定候选酶是否使用新型的荧光测定底物选择性地与肿瘤组织标本结合，而不是与正常组织结合，4）4）在动物模型中表征了细胞毒性前药的靶向酶激活的靶向酶激活。这些实验的成功完成将导致表现出新的靶向效应分子，这些分子有选择地与肿瘤结合并激活细胞毒性前药。