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The purpose of this study was to develop 64Cu-labeled trastuzumab with improved pharmacokinetics for human epidermal growth factor receptor 2 (HER2). Methods: Trastuzumab was conjugated with SCN-Bn-NOTA and radiolabeled with 64Cu. Serum stability and immunoreactivity of 64Cu-NOTA-trastuzumab were tested. Small-animal PET imaging and biodistribution studies were performed in a HER2-positive breast cancer xenograft model (BT-474). The internal dosimetry for experimental animals was determined using the image-based approach with the Monte Carlo N-particle code. Results:64Cu-NOTA-trastuzumab was prepared with high radiolabeling yield and radiochemical purity (>98%) and showed high stability in serum and good immunoreactivity. Uptake of 64Cu-NOTA-trastuzumab was highest at 48 h after injection as determined by PET imaging and biodistribution results in BT-474 tumors. The blood radioactivity concentrations of 64Cu-NOTA-trastuzumab decreased biexponentially with time in both mice with and mice without BT-474 tumor xenografts. The calculated absorbed dose of 64Cu-NOTA-trastuzumab was 0.048 mGy/MBq for the heart, 0.079 mGy/MBq for the liver, and 0.047 mGy/MBq for the spleen. Conclusion:64Cu-NOTA-trastuzumab was effectively targeted to the HER2-expressing tumor in vitro and in vivo, and it exhibited a relatively low absorbed dose due to a short residence time. Therefore, 64Cu-NOTA-trastuzumab could be applied to select the right patients and right timing for HER2 therapy, to monitor the treatment response after HER2-targeted therapy, and to detect distal or metastatic spread.

Molecular imaging includes proteomic, metabolic, cellular biologic process, and genetic imaging. In a narrow sense, molecular imaging means genetic imaging and can be called molecular-genetic imaging. Imaging reporter genes play a leading role in molecular-genetic imaging. There are 3 major methods of molecular-genetic imaging, based on optical, MRI, and nuclear medicine modalities. For each of these modalities, various reporter genes and probes have been developed, and these have resulted in successful transitions from bench to bedside applications. Each of these imaging modalities has its unique advantages and disadvantages. Fluorescent and bioluminescent optical imaging modalities are simple, less expensive, more convenient, and more user friendly than other imaging modalities. Another advantage, especially of bioluminescence imaging, is its ability to detect low levels of gene expression. MRI has the advantage of high spatial resolution, whereas nuclear medicine methods are highly sensitive and allow data from small-animal imaging studies to be translated to clinical practice. Moreover, multimodality imaging reporter genes will allow us to choose the imaging technologies that are most appropriate for the biologic problem at hand and facilitate the clinical application of reporter gene technologies. Reporter genes can be used to visualize the levels of expression of particular exogenous and endogenous genes and several intracellular biologic phenomena, including specific signal transduction pathways, nuclear receptor activities, and protein-protein interactions. This technique provides a straightforward means of monitoring tumor mass and can visualize the in vivo distributions of target cells, such as immune cells and stem cells. Molecular imaging has gradually evolved into an important tool for drug discovery and development, and transgenic mice with an imaging reporter gene can be useful during drug and stem cell therapy development. Moreover, instrumentation improvements, the identification of novel targets and genes, and imaging probe developments suggest that molecular-genetic imaging is likely to play an increasingly important role in the diagnosis and therapy of cancer.

For noninvasive monitoring of cellular status by dual reporters, a dual membrane protein reporter system was developed and its in vivo applicability was examined. Human sodium iodide symporter (hNIS) and mutant dopamine D(2) receptor (D(2)R) transgenes were chosen considering their complementarity.pIRES-hNIS/D(2)R containing NIS and D(2)R linked with an internal ribosomal entry site (IRES) was constructed and transfected into human hepatoma SK-Hep1 and rat glioma C6 cells. The cell lines stably expressing hNIS and D(2)R (named SK-ND and C6-ND) were produced, which was confirmed by messenger RNA expression of reporter genes. The functional activities of hNIS and D(2)R were measured by (125)I uptake assay and (3)H-spiperone receptor-binding assays. A biodistribution study was performed on SK-ND tumor-bearing mice using (99m)Tc-pertechnetate and (3)H-spiperone. In vivo hNIS expression was examined using (99m)Tc-pertechnetate gamma-camera imaging and, D(2)R expression was examined using a (3)H-spiperone autoradiographic study.(125)I uptake of SK-ND and C6-ND cell lines showed a maximum 97-fold and 43-fold increase, respectively, which were completely inhibited by KClO(4). Specific (3)H-spiperone binding to SK-ND and C6-ND cell homogenates was observed, which were completely inhibited by (+)-butaclamol. Among the dual reporter gene-expressing cell lines, the activities of both reporters were inversely correlated with each other. Competition assay of hNIS-expressing cells by D(2)R vector transfection and D(2)R-expressing cells by hNIS vector transfection showed a dose-dependent decrease of hNIS and D(2)R activities, respectively. In the biodistribution study, (99m)Tc-pertechnetate accumulated 10-fold and (3)H-spiperone accumulated 4-fold more in SK-ND tumors than that in parental SK tumors. In vivo imaging of (99m)Tc-pertechnetate persisted until 5 wk after the cell graft in SK-ND tumors. Autoradiographic study of brain tissues from these mice also revealed an accumulation of (3)H-spiperone in SK-ND tumors.We developed a dual membrane-bound positron and gamma-imaging reporter system of hNIS and D(2)R. We observed its reporting capability in vitro and in vivo and elucidated that these 2 membrane protein reporters competed with each other in their expression. Although we expect that hNIS and D(2)R transgenes can complement each other as a dual reporter system, we suggest that one needs to validate the ratio of expression of the 2 membrane protein reporter transgenes for cellular status tracking.