Non-Immunogenic ADEPT: Human Enzymes & Delivery Vehicles

描述(由申请人提供)：到目前为止，抗体导向酶前药物疗法(ADEPT)治疗患者使用的是非人类来源的酶。这些外来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两种人类酶的新方法。具体地说，我们将使用人胸苷磷酸化酶(HTP)，目前它是前药物治疗的靶点，因为它在一些人类肿瘤中过度表达，我们假设通过将额外的HTP靶向肿瘤，我们将能够使用它的前药5‘-脱氧-5-氟尿苷。作为一种更有效的抗肿瘤疗法。虽然人类嘌呤核苷磷酸化酶(HPNP)的广泛表达使其不能直接用作ADEPT的酶，但我们假设我们可以产生一种底物专一性改变的突变体，它可以使用含有前药的腺苷和脱氧腺苷作为底物。利用同一抗体可以输送不同分子的递送系统，可以很容易地评估许多不同的潜在治疗性蛋白质的疗效。我们假设我们可以生产一种由人中性粒细胞弹性蛋白酶(NE)及其抑制物(NEI)组成的非免疫原性通用递送系统。这些都形成了一个强大而稳定的复合体，我们假设NE/NEI相互作用可以用来为ADEPT制作抗体/酶复合体。具体地说，hTP和突变体hPNP将通过柔性接头序列连接到NE基因的3‘端表达，NEI将通过柔性接头序列连接到肿瘤相关抗原特异性抗体的重链3’端，并以适当的轻链表达。或者，它可以与更小的抗体片段融合，如ScFv、Fab和F(Ab2‘)。如果我们在使用NE/NEI系统时遇到困难，我们会使用“S.tag/S.Protein”系统。这些酶和融合蛋白将在体外评估它们将前药物转化为对培养的癌细胞有效的细胞毒剂的能力。如果观察到疗效，我们将在小鼠身上评估这些蛋白质。将确定融合蛋白和前药的最大耐受量。生物分布、药代动力学和肿瘤靶向将通过使用125I标记蛋白质的传统技术和使用124I标记蛋白质的高分辨率小动物PET成像进行评估。针对人类CEA、HER2/neu和TFR的抗体将被比较其靶向表达这些抗原的肿瘤的能力。携带肿瘤的小鼠将由ADEPT使用最有效的抗体/酶组合进行治疗(S)。我们假设，我们将能够开发出一种在小鼠身上有效的治疗方法，这种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.