Targeted Enzymes for Prodrug Therapy

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，其将前药转化为毒素用于癌症治疗。这种方法的基本原理是基于已发表的单链抗体（scFv）靶向酶的实验，这些酶特异性激活强效细胞毒性剂的前药。这些前药的细胞毒性显著低于通过用靶向酶裂解释放的毒素。我们假设，当针对从手术切除后的患者分离的肿瘤细胞淘选时，具有直接构建到蛋白质支架中的靶向部分的噬菌体酶将含有肿瘤选择性效应分子。在新鲜人肿瘤细胞上淘选酶文库的益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库，以及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的能力。具体目的是1）在噬菌体上产生酶的多样性随机环文库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的噬菌体展示的新方法鉴定选择性结合肿瘤细胞的噬菌体酶，3）使用新的荧光底物确定候选酶是否选择性结合肿瘤组织样本而不结合正常组织，和4）在动物模型中体外和体内表征细胞毒性前药的靶向酶活化。这些实验的成功完成将导致新的靶向效应分子，选择性地结合到肿瘤和激活细胞毒性前药的证明。

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.