Non-Immunogenic ADEPT: Human Enzymes & Delivery Vehicles

描述（由申请人提供）：迄今为止，针对患者的抗体导向酶前药物治疗（ADEPT）使用了非人类来源的酶。这些外源蛋白的免疫原性使其无法长期用于治疗。为了生产免疫原性降低的酶，我们将尝试开发使用2种人类酶的新方法。具体来说，我们将使用人类胸苷磷酸化酶（hTP），目前是前药治疗的靶标，因为它在一些人类肿瘤中过表达，我们假设通过靶向肿瘤额外的hTP，我们将能够使用它的前药，5'-脱氧-5-氟吡啶。作为一种更有效的抗肿瘤治疗手段。虽然广泛的表达人类嘌呤核苷磷酸化酶(hPNP)排除了其直接使用的酶熟练我们假设我们可以产生一个突变改变了底物特异性,可以使用腺苷和脱氧腺苷含有高活性化合物作为底物。一种可以使用相同抗体递送不同分子的递送系统使得评估许多不同潜在治疗蛋白的功效成为可能。我们假设我们可以制造一个由人中性粒细胞弹性酶（NE）及其抑制剂（NEI）组成的非免疫原性通用递送系统。这些形成了一个强而稳定的复合物，我们假设NE/NEI相互作用可以用来制造ADEPT的抗体/酶复合物。具体来说，hTP和突变型hPNP将通过柔性连接子序列连接到NE基因的3‘端，NEI将通过柔性连接子序列连接到肿瘤相关抗原特异性抗体重链的3’端，并通过适当的轻链表达。或者，它可以融合到更小的抗体片段，如scFv、Fab和F（ab2’）。如果我们在使用NE/NEI系统时遇到困难，我们将使用“S.tag/S”。蛋白质”系统。这些酶和融合蛋白将在体外评估它们将前药转化为对培养癌细胞有效的细胞毒性药物的能力。如果观察到疗效，我们将在小鼠中评估蛋白质。融合蛋白和前药的最大耐受剂量将被确定。生物分布、药代动力学和肿瘤靶向性将通过使用125I标记蛋白的传统技术和使用124i标记蛋白的高分辨率小动物PET成像进行评估。将比较人CEA、HER2/neu和TfR特异性抗体对表达这些抗原的肿瘤的靶向能力。用最有效的抗体/酶组合治疗荷瘤小鼠。我们假设我们将能够开发一种对小鼠有效的治疗方法，可以很容易地应用于治疗人类恶性肿瘤。

DESCRIPTION (provided by applicant): To date, antibody-directed enzyme prodrug therapy (ADEPT) treatment of patients has used enzymes of nonhuman origin. The immunogenicity of these foreign proteins precludes their long-term use for therapy. To produce enzymes of decreased immunogenicity, we will attempt to develop novel approaches using 2 human enzymes. Specifically, we will use human thymidine phosphorylase (hTP), currently a target of prodrug therapy because it is overexpressed in some human tumors, and we hypothesize that by targeting additional hTP to tumors, we will be able to use its prodrug, 5'-deoxy-5-fluorouridine. as a more effective anti-tumor therapeutic. Although the wide-spread expression of human purine nucleoside phosphorylase (hPNP) precludes its direct use as an enzyme for ADEPT we hypothesize that we can produce a mutant with altered substrate specificity that can use adenosine and deoxyadenosine containing prodrugs as substrates. A delivery system in which the same antibody can be used to deliver different molecules makes it possible to readily evaluate the efficacy of many different potential therapeutic proteins. We hypothesize that we can produce a nonimmunogenic universal delivery system comprised of human neutrophil elastase (NE) and its inhibitor (NEI). These form a strong and stable complex and we hypothesize that the NE/NEI interaction can be used to make antibody/enzyme complexes for ADEPT. Specifically, hTP and mutant hPNP will be expressed connected to the 3' end of the NE gene via a flexible linker sequence and NEI will be attached, via a flexible linker sequence, to the 3' end of the heavy chain from an antibody specific for a tumor associated antigen and expressed with the appropriate light chain. Alternatively, it can be fused to smaller antibody fragments such as scFv, Fab, and F(ab2'). If we encounter difficulties with the NE/NEI system, we will use the "S.tag/S.protein" system. The enzymes and fusion proteins will be evaluated in vitro for their ability to convert prodrugs to cytotoxic agents effective against cultured cancer cells. If efficacy is observed, we will evaluate the proteins in mice. The maximum tolerated dose for the fusion proteins and the prodrugs will be determined. Biodistribution, pharmacokinetics and tumor targeting will be evaluated by traditional techniques using 125I labeled proteins and by high-resolution small animal PET imaging with 124I-labeled proteins. Antibodies specific for human CEA, HER2/neu and TfR will be compared for their ability to target tumors expressing these antigens. Mice bearing tumors will be treated by ADEPT using the most effective antibody/enzyme combination(s). We hypothesize that we will be able to develop a therapeutic approach effective in mice that can readily be applied to the treatment of human malignancy.