Recombinant Carcinoembronic Antigen as PET Reporter Gene

报告基因技术为基因基础研究提供了重要的窗口 用于细胞培养系统中的基因表达，以及转基因细胞或动物中基因表达的活体测量。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转基因免疫效应细胞。目前的报告基因成像系统在临床上的应用受到外源基因潜在的免疫原性、生物活性报告蛋白的毒性、检测的敏感性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，如跟踪转基因T细胞。CEA应该是非免疫原性的，因为它是人类自身抗原。正常成人的内源性组织表达非常有限，免疫细胞中不存在内源性组织表达，包括B和T淋巴细胞。我们已经开发了两种新型的CEA特异性PET示踪剂--被称为“mini Body”和“Diabody”的基因工程抗体片段--它们已经通过microPET在小鼠模型中进行了评估，目前正在进行临床研究。 用正电子发射体124I或64Cu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)我们将构建和评估跨膜锚定和内化形式的CEA作为报告基因。这种天然CEA的激发是必需的，因为CEA通过GPI连接与细胞膜相连，并且可以被释放和脱落。稳定转染的淋巴样细胞株将被 评估抗CEA抗体的结合和内化情况。(2)CEA报告基因将用PET同位素标记的抗CEA小体和小体进行体内显像。将在携带稳定转基因异种移植的小鼠身上进行初步鉴定。还将开发逆转录病毒载体，并通过体内成像对小鼠初级T细胞的转导和监测进行评估。(3)在噬菌体展示法分离的CEA结合肽的基础上，开发一种新型的PET报告分子探针。在项目结束时，将对CEA PET报告基因以及抗CEA PET报告基因的设计和功能进行深入的探索和优化。该系统将为监测基因表达和追踪患者体内修饰的免疫细胞提供一种强大的、非侵入性的方法。

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.