Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusion: The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo.

Fig. 1

Schematic representation of (a) the CEA reporter gene construct, including the restriction enzyme cut sites. ECD = extracellular domain; TMD = transmembrane domain. (b) positioning of the reporter protein (NA3-FcγRIIb) in the cellular architecture and its interaction with the ¹²⁴I-labeled reporter probe (anti-CEA scFv-Fc antibody fragment).

Fig. 2

NA3-FcγRIIb reporter gene expression in Jurkat T cells.

Fig. 3

Western blot of N-A3 expression in NA3-FcγRIIb transfected cells (a) Blot probed with anti-CEA cT84.66 primary and AP-conjugated anti-human Fcγ-specific secondary antibodies; (b) Loading control blot, probed with anti-β actin mouse and AP-conjugated anti-mouse Fcγ-specific antibodies. Lane 1: High N-A3 expression; Lane 2: Medium N-A3 expression; Lane 3: Low N-A3 expression; Lane 4: Molecular weight standard; Lane 5: LS174T (CEA +) control; Lane 6: Non-transfected Jurkat (−) control.

Fig. 4

Immunohistochemistry of N-A3/CEA expression in tumors (40x magnification). Parental Jurkat and LS174T tumors are included as negative and positive controls.

Fig. 5

MicroPET/microCT imaging of (a) High; (b) Medium; and (c) Low N-A3 expressing Jurkat xenografts, using ¹²⁴I-labeled anti-CEA scFv-Fc H310A antibody fragment at 4, 20 and 48h post tracer injection. (+) and (−) indicates transfected and control Jurkat tumors, respectively.