Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging
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Reporter genes can provide a way of noninvasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression, or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression.
To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human FcγRIIb receptor. The NA3-FcγRIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging.
Flow cytometry identified Jurkat clones stably expressing NA3-FcγRIIb at low, medium, and high levels. High and medium NA3-FcγRIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA 124I-labeled single-chain Fv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4 h. MicroPET image quantitation showed tumor activity of 1.8 ± 0.2, 15.2 ± 1.3, and 4.6 ± 1.2 percent injected dose per gram (%ID/g) at 4, 20, and 48 h, respectively. Biodistribution at 48 h demonstrated tumor uptake of 4.8 ± 0.8%ID/g.
The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo.
KeywordsT cells CEA reporter gene Jurkat xenograft 124I-labeled scFv-Fc antibody fragment MicroPET/microCT imaging
We thank the UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core facility. We are especially grateful to Dr. David Stout, Waldemar Ladno, and Judy Edwards at the University of California Los Angeles for their assistance with the microPET/CT scans. We also acknowledge the assistance of Sofia Loera at the City of Hope Comprehensive Cancer Center Anatomic Pathology Core Facility for performing the immunohistochemistry work. This work was supported by P50 CA 086306; National Institute of Health (NIH) grants CA 043904 and CA 086306; Department of Defense grants DAMD 17-00-1-203 and DAMD 17-00-1-0150. A.M.W., A.C., H.H., and J.B. are members of the UCLA Jonsson Comprehensive Cancer Center (NIH CA 016042).
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