Non-Immunogenic ADEPT: Human Enzymes & Delivery Vehicles

描述（由申请方提供）：迄今为止，患者的抗体导向酶前药治疗（ADEPT）使用非人源酶。这些外源蛋白的免疫原性排除了它们长期用于治疗。为了生产免疫原性降低的酶，我们将尝试使用2种人酶开发新方法。具体地说，我们将使用人胸苷磷酸化酶（hTP），目前是前药治疗的靶点，因为它在一些人肿瘤中过表达，我们假设通过将额外的hTP靶向肿瘤，我们将能够使用其前药，5 '-脱氧-5-氟尿苷。作为一种更有效的抗肿瘤治疗方法。虽然人嘌呤核苷磷酸化酶（hPNP）的广泛表达排除了其直接用作ADEPT的酶，但我们假设我们可以产生具有改变的底物特异性的突变体，其可以使用含有腺苷和脱氧腺苷的前药作为底物。其中相同抗体可用于递送不同分子的递送系统使得可以容易地评估许多不同的潜在治疗性蛋白质的功效。我们假设，我们可以产生一个非免疫原性的通用传递系统组成的人中性粒细胞弹性蛋白酶（NE）及其抑制剂（NEI）。这些形成了一个强大的和稳定的复合物，我们假设NE/NEI相互作用可用于使抗体/酶复合物的ADEPT。具体地，hTP和突变型hPNP将通过柔性接头序列连接到NE基因的3'末端来表达，并且NEI将通过柔性接头序列连接到来自对肿瘤相关抗原特异性的抗体的重链的3'末端，并且与适当的轻链一起表达。或者，它可以与较小的抗体片段如scFv、Fab和F（ab 2 '）融合。如果我们在NE/NEI系统中遇到困难，我们将使用“S.tag/S.protein”系统。将在体外评价酶和融合蛋白将前药转化为有效对抗培养的癌细胞的细胞毒性剂的能力。如果观察到疗效，我们将在小鼠中评估蛋白质。将确定融合蛋白和前药的最大耐受剂量。生物分布、药代动力学和肿瘤靶向将通过使用125 I标记蛋白质的传统技术和使用124 I标记蛋白质的高分辨率小动物PET成像进行评价。将比较人CEA、HER 2/neu和TfR特异性抗体靶向表达这些抗原的肿瘤的能力。将使用最有效的抗体/酶组合通过ADEPT治疗荷瘤小鼠。我们假设，我们将能够开发一种在小鼠中有效的治疗方法，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.