Targeted Enzymes for Prodrug Therapy

描述(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，这种酶能特异性地激活有效的细胞毒剂的前体药物。这些前药的细胞毒性明显低于目标酶裂解释放的毒素。我们推测，当直接构建到蛋白支架中的带有靶向部分的噬菌体酶对抗手术切除后患者分离的肿瘤细胞时，将含有肿瘤选择性效应分子。在新鲜的人类肿瘤细胞上平移酶文库的好处包括存在大量可能的靶点，靶点将处于其原始配置，减少了与正常组织结合的配位体池，以及能够使用不断增加的前体药物组合快速测试这些新型靶向试剂的有效性。具体目标是1)在肿瘤组织上生成不同的随机环库2)手术样本肿瘤组织，分离少量新鲜的肿瘤细胞，并使用一种新的基于PCR的噬菌体展示方法鉴定已选择性结合到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地结合到肿瘤组织标本上，以及4)在动物模型中表征细胞毒性前体药物的靶向性酶激活。这些实验的成功完成将导致新的靶向效应分子的展示，这些分子选择性地与肿瘤结合并激活细胞毒前药。

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.