Recombinant Carcinoembronic Antigen as PET Reporter Gene

报告基因技术为基因基础研究提供了一个重要窗口 用于细胞培养系统中的基因表达，以及用于遗传修饰的细胞或动物中的基因表达的体内测量。报道基因方法在临床环境中也越来越重要，以监测治疗基因的表达并在癌症的基因治疗或免疫治疗中追踪遗传修饰的免疫效应细胞。目前的报告基因成像系统在临床环境中的应用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质，癌胚抗原（CEA）作为PET报告基因，用于人类应用，如跟踪遗传修饰的T细胞。CEA应该是非免疫原性的，因为它是人自身抗原。内源性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。我们已经开发了两种新的CEA特异性PET示踪剂-基因工程抗体片段，称为“minibody”和“diabody”-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。 在用正电子发射体124 I或64 Cu进行放射性标记后，这些示踪剂在鼠异种移植模型中达到10- 25%注射剂量/克。(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。天然CEA的这种基序化是必需的，因为CEA通过GPI连接与细胞膜缔合，并且可以被释放和脱落。将稳定转染的淋巴细胞系 评价抗CEA抗体结合和内化。(2)CEA报告基因将通过使用PET同位素标记的抗CEA微抗体和双抗体的体内成像来评估。将在携带稳定转染异种移植物的小鼠中进行初始表征。将开发逆转录病毒载体，还将评价通过体内成像对鼠原代T细胞的转导和监测。(3)一类新的PET报告探针将开发，基于CEA结合肽先前分离的噬菌体展示。在项目结束时，CEA PET报告基因以及抗CEA PET报告探针的设计和功能将被彻底探索和优化。该系统将提供一种强大的非侵入性方法，用于监测患者的基因表达和跟踪修饰的免疫细胞。

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.