Recombinant Carcinoembronic Antigen as PET Reporter Gene

重组癌胚抗原作为 PET 报告基因

• 批准号: 7039880
• 负责人: Anna M Wu
• 金额: $ 16.25万
• 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: 2005-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7039880
• 关键词: T lymphocyte; antigen antibody reaction; bioimaging /biomedical imaging; biotechnology; carcinoembryonal antigen; gene expression; image enhancement; immune response; laboratory mouse; positron emission tomography; recombinant proteins; reporter genes; transfection

Reporter gene technology has provided an important window for basic investigations of gene expression in cell culture systems, and for in vivo measurement of gene expression in genetically modified cells or animals. Reporter gene approaches are gaining in importance in clinical settings as well, to monitor expression of therapeutic genes and to track genetically modified immune effector cells in gene therapy or immunotherapy of cancer. The translation of current reporter gene imaging systems into the clinical setting is limited by potential immunogenicity of foreign genes, toxicity of biologically active reporter proteins, sensitivity of detection, and background signal issues. We propose to develop an endogenous human protein, carcinoembryonic antigen (CEA) as a PET reporter gene for human applications such as tracking genetically modified T cells. CEA should be nonimmunogenic since it is human self-antigen. Endogenous tissue expression is very limited in normal adults and non-existent in immune cells, including B and T lymphocytes. We have developed two novel CEA-specific PET tracers - genetically engineered antibody fragments called the "minibody" and "diabody" - which have been evaluated in mouse models by microPET and which are currently in clinical studies. Following radiolabeling with the positron emitters 124I or 64Cu, these tracers reach 10-25 % injected dose per gram in murine xenograft models. (1) We will construct and evaluate transmembrane-anchored and internalizing forms of CEA for use as reporter genes. This motidication of native CEA is required because CEA is associated with the cell membrane via a GPI linkage, and can be released and shed. Stably transfected lymphoid cell lines will be evaluated for anti-CEA antibody binding and internalization. (2) CEA reporter genes will be evaluated by in vivo imaging using PET isotope labeled anti-CEA minibodies and diabodies. Initial characterization will be carried out in mice bearing stably transfected xenografts. Retroviral vectors will be developed and transduction and monitoring of murine primary T cells by imaging in vivo will also be evaluated. (3) A new class of PET reporter probe will be developed, based on CEA-binding peptides previously isolated by phage display. At the end of the project, design and function of the CEA PET reporter gene as well as anti-CEA PET reporter probes will be thoroughly explored and optimized. This system should provide a powerful, non-invasive method for monitoring gene expression and tracking modified immune cells in patients.

报告基因技术为基因基础研究提供了重要窗口 细胞培养系统中的表达，以及转基因细胞或动物中基因表达的体内测量。报告基因方法在临床环境中也变得越来越重要，用于监测治疗基因的表达并在癌症的基因治疗或免疫治疗中跟踪基因修饰的免疫效应细胞。当前报告基因成像系统向临床环境的转化受到外源基因的潜在免疫原性、生物活性报告蛋白的毒性、检测灵敏度和背景信号问题的限制。我们建议开发一种内源性人类蛋白——癌胚抗原（CEA）作为 PET 报告基因，用于人类应用，例如追踪转基因 T 细胞。 CEA 应该是非免疫原性的，因为它是人类自身抗原。内源组织表达在正常成人中非常有限，并且在免疫细胞（包括 B 和 T 淋巴细胞）中不存在。我们开发了两种新型 CEA 特异性 PET 示踪剂 - 基因工程抗体片段，称为“微型抗体”和“双抗体” - 已通过 microPET 在小鼠模型中进行了评估，目前正在进行临床研究。 在用正电子发射体 124I 或 64Cu 进行放射性标记后，这些示踪剂在小鼠异种移植模型中达到每克注射剂量的 10-25%。 (1) 我们将构建并评估 CEA 的跨膜锚定和内化形式，以用作报告基因。天然 CEA 的这种刺激是必需的，因为 CEA 通过 GPI 连接与细胞膜相关联，并且可以释放和脱落。稳定转染的淋巴细胞系 评估抗 CEA 抗体结合和内化。 (2) CEA报告基因将使用PET同位素标记的抗CEA微型抗体和双抗体通过体内成像进行评估。初步表征将在携带稳定转染异种移植物的小鼠中进行。将开发逆转录病毒载体，并通过体内成像对小鼠原代 T 细胞的转导和监测进行评估。 (3) 基于先前通过噬菌体展示分离的 CEA 结合肽，将开发一类新的 PET 报告探针。项目结束时，将彻底探索和优化CEA PET报告基因以及抗CEA PET报告基因探针的设计和功能。该系统应该提供一种强大的、非侵入性的方法来监测基因表达和跟踪患者体内修饰的免疫细胞。