Non-Immunogenic ADEPT: Human Enzymes & Delivery Vehicles

非免疫原性 ADEPT：人类酶

基本信息

批准号：
6908793
负责人：
Sherie L Morrison
金额：
$ 26.89万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-01 至 2009-03-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：迄今为止，抗体导向的酶前药疗法（ADEPT）对患者的治疗已使用非人类来源的酶。这些外源蛋白的免疫原性妨碍了它们的长期治疗用途。为了生产免疫原性降低的酶，我们将尝试使用 2 种人类酶开发新方法。具体来说，我们将使用人胸苷磷酸化酶（hTP），它目前是前药治疗的靶标，因为它在一些人类肿瘤中过度表达，并且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。作为更有效的抗肿瘤治疗方法。尽管人嘌呤核苷磷酸化酶 (hPNP) 的广泛表达妨碍了其直接用作 ADEPT 的酶，但我们假设我们可以产生底物特异性改变的突变体，该突变体可以使用含有腺苷和脱氧腺苷的前药作为底物。可以使用相同的抗体来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。我们假设我们可以生产一种由人中性粒细胞弹性蛋白酶（NE）及其抑制剂（NEI）组成的非免疫原性通用递送系统。它们形成强大而稳定的复合物，我们假设 NE/NEI 相互作用可用于制备 ADEPT 的抗体/酶复合物。具体地，hTP和突变体hPNP将通过柔性接头序列连接至NE基因的3'端来表达，并且NEI将通过柔性接头序列连接至来自对以下抗体具有特异性的重链的3'端：肿瘤相关抗原并用适当的轻链表达。或者，它可以与较小的抗体片段融合，例如 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. Protein”系统。将在体外评估酶和融合蛋白将前药转化为有效对抗培养癌细胞的细胞毒剂的能力。如果观察到功效，我们将评估小鼠体内的蛋白质。将确定融合蛋白和前药的最大耐受剂量。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 成像来评估生物分布、药代动力学和肿瘤靶向。将比较人类 CEA、HER2/neu 和 TfR 特异性抗体靶向表达这些抗原的肿瘤的能力。携带肿瘤的小鼠将通过 ADEPT 使用最有效的抗体/酶组合进行治疗。我们假设我们将能够开发出一种对小鼠有效的治疗方法，并且可以很容易地应用于人类恶性肿瘤的治疗。