Non-Immunogenic ADEPT: Human Enzymes & Delivery Vehicles

非免疫原性 ADEPT：人类酶

• 批准号: 7218077
• 负责人: Sherie L Morrison
• 金额: $ 23.44万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/7218077
• 关键词: 2' -deoxyadenosine; 6-methylpurine; Adenine; Adenosine; Amino Acids; Animals; Antibodies; Antibody-Directed Enzyme Prodrug Therapy; Antigens; Avidin; Bacteria; Biodistribution; Biotin; Blood Circulation; Chickens; Chimeric Proteins; Complex; Cytotoxic agent; Deoxyadenosines; Doxifluridine; Drug Kinetics; ERBB2 gene; Endoribonucleases; Enzyme Gene; Enzymes; Exhibits; Fludarabine phosphate; Genes; Human; Immune response; Immunoglobulin Fragments; In Vitro; Kazal Pancreatic Trypsin Secretory Inhibitor; Label; Leukocyte Elastase; Light; Malignant Neoplasms; Mammalian Cell; Maximum Tolerated Dose; Monitor; Multienzyme Complexes; Mus; No Evidence of Disease; Pancreatic ribonuclease; Patients; Penetration; Positron-Emission Tomography; Principal Investigator; Prodrugs; Protein Overexpression; Proteins; Protocols documentation; Purine-Nucleoside Phosphorylase; Residual Neoplasm; Resolution; Ribonucleases; Risk; Substrate Specificity; System; Techniques; Therapeutic; Thymidine Phosphorylase; Time; Toxic effect; Tumor Antigens; base; cancer cell; clinical application; deoxyadenosine; immunogenic; immunogenicity; in vivo; inhibitor/antagonist; mutant; novel; novel strategies; programs; size; therapeutic protein; trypsin-like serine protease; tumor

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，抗体定向的酶前药治疗（Adept）治疗患者已经使用了非人类来源的酶。这些异物蛋白质的免疫原性无法长期用于治疗。为了产生降低免疫原性的酶，我们将尝试使用2种人类酶开发新的方法。具体而言，我们将使用人类胸苷磷酸化酶（HTP），目前是前药治疗的靶标，因为它在某些人类肿瘤中过表达，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'-Deoxy-5-5-5-5-5-氟尿嘧啶。作为一种更有效的抗肿瘤治疗。尽管人类嘌呤核苷磷酸化酶（HPNP）的广泛表达排除了其作为熟练的酶的直接用途，但我们假设我们可以产生具有改变底物特异性的突变体，可以使用腺苷和含有脱氧腺苷的含有药物的药物剂。相同抗体可用于输送不同分子的递送系统，可以轻松评估许多潜在的治疗蛋白的功效。我们假设我们可以产生由人类嗜中性粒细胞弹性酶（NE）及其抑制剂（NEI）组成的非免疫原性递送系统。这些形成强大而稳定的复合物，我们假设NE/NEI相互作用可用于使抗体/酶复合物用于熟练。具体而言，HTP和突变的HPNP将通过柔性接头序列表示与NE基因的3'端连接到NE基因的3'端，NEI将通过柔性接头序列连接到重链的3'末端，从肿瘤相关的抗体特异性抗体的3'端，并与适当的轻型链表示。另外，它可以融合到较小的抗体片段，例如SCFV，FAB和F（AB2'）。如果我们在NE/NEI系统方面遇到困难，我们将使用“ s.tag/s. protein”系统。将在体外评估酶和融合蛋白，以使其能够将前药转化为有效针对培养的癌细胞的细胞毒性剂。如果观察到功效，我们将评估小鼠的蛋白质。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传统技术使用125i标记的蛋白质以及具有124i标记蛋白的高分辨率小动物宠物成像进行评估。将比较针对人CEA，HER2/NEU和TFR的抗体，以靶向表达这些抗原的肿瘤的能力。使用最有效的抗体/酶组合（S），将通过熟练治疗轴承肿瘤的小鼠。我们假设我们将能够在小鼠中开发一种有效的治疗方法，该方法很容易应用于治疗人类恶性肿瘤。