Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging

Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, 700 Westwood Plaza, Los Angeles, CA 90095, USA.
European Journal of Nuclear Medicine (Impact Factor: 5.38). 09/2008; 36(1):104-14. DOI: 10.1007/s00259-008-0921-z
Source: PubMed


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 Fc gamma RIIb receptor. The NA3-Fc gamma RIIb 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-Fc gamma RIIb at low, medium, and high levels. High and medium NA3-Fc gamma RIIb 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 (124)I-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.

Download full-text


Available from: Jonathan Braun
  • Source
    • "Reporter-positive tumors showed clear signal at 24 h (Fig. 5), and the tumor-to-background ratio, determined by ROI analysis of the images, was 2.8. In our previous study with the noninternalizing NA3-FcγRIIb reporter gene, we determined tumor-to-background ratio of 6.1 at 20 h in tumors, showing stronger IHC staining and expressing about 25,000 reporters per cell [9]. This observation suggests that even tumor masses with lower than 25,000 surface reporters (i.e., TR(1–99)-NA3 transfected Jurkat tumors) can still be detected by PET. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo. The transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of ¹²⁵I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using ¹²⁴I- or ⁶⁴Cu-scFv-Fc as tracers. Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1-99)-NA3 stained weakly. Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice. The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.
    Full-text · Article · Jun 2011 · Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging
  • [Show abstract] [Hide abstract]
    ABSTRACT: Antibodies have attained a central role as targeted therapeutics, with several significant drugs on the market and many more in clinical development for oncological applications. Expansion of the role of antibodies in cancer imaging has been accelerated by a number of factors, including the recognition that antibodies can provide a powerful class of molecular imaging probes for interrogating cell surfaces in vivo. Identification of relevant cell surface biomarkers as imaging targets, coupled with advances in antibody technology, facilitate the generation of antibodies optimized for noninvasive imaging. Developments in imaging instrumentation and radionuclide availability have paved the way for broader evaluation and implementation of radioimmunoscintigraphy and immunoPET. Antibody imaging can provide a sensitive, noninvasive means for molecular characterization of cell surface phenotype in vivo, which can in turn guide diagnosis, prognosis, therapy selection, and monitoring of treatment in cancer.
    No preview · Article · Jun 2008 · The Cancer Journal
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.
    Preview · Article · Apr 2012 · Theranostics
Show more