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.
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