Targeted Enzymes for Prodrug Therapy

描述（由申请人提供）：利用噬菌体展示的随机酶库，我们的目标是开发肿瘤靶向效应分子，将前药转化为癌症治疗的毒素。这种方法的基本原理是基于已发表的单链抗体（scFv）靶向酶的实验，该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与目标酶裂解释放的毒素。我们假设，当从手术切除后的患者中分离出肿瘤细胞时，具有靶向部分的噬菌体酶直接构建在蛋白质支架中，将含有肿瘤选择性效应分子。在新鲜的人类肿瘤细胞上筛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，减少与正常组织结合的配体池，以及使用不断增长的前药组合快速测试这些新型靶向药物的功效的能力。具体目标是：1)在噬菌体上生成多种随机循环酶文库；2)手术取样肿瘤组织，分离少量新鲜肿瘤细胞，并使用基于pcr的噬菌体展示新方法鉴定选择性结合肿瘤细胞的噬菌体酶；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标本，而不是正常组织。4)在体外和体内动物模型中表征细胞毒性前药的靶向酶激活。这些实验的成功完成将导致新的靶向效应分子的展示，这些分子选择性地结合肿瘤并激活细胞毒性前药。

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.