Imaging of EGFR and EGFR tyrosine kinase overexpression in tumors by nuclear medicine modalities.

Protein tyrosine kinases (PTKs) play a pivotal role in signal transduction pathways and in the development and maintenance of various cancers. They are involved in multiple processes such as transcription, cell cycle progression, proliferation, angiogenesis and inhibition of apoptosis. Among the PTKs, the EGFR is one of the most widely studied and has emerged as a promising key target for the treatment of cancer. Indeed, several drugs directed at this receptor are FDA-approved and many others are at various stages of development. However, thus far, the therapeutic outcome of EGFR-targeted therapy is suboptimal and needs to be refined. Quantitative PET molecular imaging coupled with selective labelled biomarkers may facilitate in vivo EGFR-targeted drug efficacy by noninvasively assessing the expression of EGFR in tumor, guiding dose and regime by measuring target drug binding and receptor occupancy as well as potentially detecting the existence of a primary or secondary mutation leading to either drug interaction or failure of EGFR recognition by the drug. This review describes the attempts to develop labelled EGFR molecular imaging agents that are based either on low molecular weight tyrosine kinase inhibitors or monoclonal antibodies directed to the extracellular binding domain of the receptor to be used in nuclear medicine modalities.

Preparation and biological evaluation of 64Cu-CB-TE2A-sst2-ANT, a somatostatin antagonist for PET imaging of somatostatin receptor-positive tumors.

UNLABELLED: Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst2-ANT (64Cu-CB-TE2A-sst2-ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors. METHODS: Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64Cu-CB-TE2A-sst2-ANT was compared with that of the 64Cu-labeled agonist 64Cu-CB-TE2A-tyrosine3-octreotate (64Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and small-animal PET/CT of 64Cu-CB-TE2A-sst2-ANT was performed. RESULTS: The dissociation constant value for the radiopharmaceutical was determined to be 26 +/- 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64Cu-CB-TE2A-sst2-ANT showed significantly less internalization than did 64Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64Cu-CB-TE2A-sst2-ANT from the blood was rapid, whereas the clearance of 64Cu-CB-TE2A-sst2-ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64Cu-CB-TE2A-sst2-ANT than those for 64Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64Cu-CB-TE2A-sst2-ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h. CONCLUSION: The PET radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptor-positive tumors.

Correlating EGFR expression with receptor-binding properties and internalization of 64Cu-DOTA-cetuximab in 5 cervical cancer cell lines.

UNLABELLED: The anti-epidermal growth factor receptor (anti-EGFR) antibody cetuximab is clinically approved for the treatment of EGFR-expressing metastatic colorectal cancer and advanced head and neck cancer. Overexpression of EGFR has also been found in more than 70% of carcinomas of the cervix. The overall goal of this study was to determine whether (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-cetuximab has potential as an agent for measuring EGFR concentration by PET imaging in cervical cancer tumors. METHODS: Cetuximab was conjugated to the bifunctional chelator DOTA and labeled with (64)Cu. EGFR messenger RNA (mRNA) expression was correlated with EGFR densities on the cell surface of 5 different cervical cancer cell lines and with receptor function, measured by internalization of (64)Cu-DOTA-cetuximab. Imaging in tumor-bearing mice with small-animal PET was performed using the highest-expressing cervical cancer cell line. RESULTS: The affinity of (64)Cu-DOTA-cetuximab binding for the EGFR was similar in 4 EGFR-positive lines, varying from 0.1 to 0.7 nM. The mRNA expression corresponded well with EGFR densities and levels of internalization, with responses decreasing in the order of CaSki>ME-180>DoTc2 4510>HeLa>C-33A. Biodistribution and small-animal PET studies with (64)Cu-DOTA-cetuximab in CaSki tumor-bearing nude mice showed relatively high tumor uptake at 24 h after injection (13.2+/-1.2 percentage of injected activity per gram), although there was also significant retention of activity in the blood and liver accumulation. CONCLUSION: (64)Cu-DOTA-cetuximab was successfully used to detect and quantify EGFR expression in cervical cancer tumors, and small-animal PET/CT of EGFR-expressing CaSki tumors suggests potential for PET/CT of EGFR-positive tumors.

The role of p53 in the trafficking of copper-64 to tumor cell nuclei.

Copper-64 (T(1/2) = 12.7 h; beta(+): 17.8%, beta(-): 41%) has applications in both positron emission tomography (PET) imaging and targeted radiotherapy of cancer. Copper-64 radiopharmaceuticals have shown tumor growth inhibition with a relatively low radiation dose in animal models; however, the mechanism of cytotoxicity has not been fully elucidated. Here, we report an investigation on the potential role of the tumor suppressor protein p53 in trafficking (64)Cu to tumor cell nuclei. Two EGFR expressing human colorectal cell lines (HCT 116 +/+ and HCT 116 -/-) that are positive or negative for p53 expression respectively, were used to compare internalization and nuclear localization of [(64)Cu]copper acetate and of (64)Cu-DOTA-cetuximab, a monoclonal anti-EGFR antibody. [(64)Cu]copper acetate uptake into cells was similar between the two cell lines during a 24 h time course. In contrast, the uptake of [(64)Cu]copper acetate in the nuclei of HCT 116 +/+ cells was significantly higher than in HCT 116 -/- cells (p < 0.0001) at 24 h. There was no difference in receptor binding, receptor-mediated internalization, and efflux of (64)Cu-DOTA-cetuximab between the two HCT 116 cells lines. However, nuclear localization of (64)Cu-DOTA-cetuximab showed increased uptake in the nuclei of HCT 116 +/+ cells as early as 4 h. These data demonstrate that (64)Cu is delivered to tumor cell nuclei in a p53 positive cell line in significantly greater amounts than in p53 negative cells by both non-specific and receptor-mediated uptake mechanisms.

Nuclear uptake and dosimetry of 64Cu-labeled chelator somatostatin conjugates in an SSTr2-transfected human tumor cell line.

UNLABELLED: 64Cu radiopharmaceuticals have shown tumor growth inhibition in tumor-bearing animal models with a relatively low radiation dose that may be related to nuclear localization of the 64Cu in tumor cells. Here we address whether the nuclear localization of 64Cu from a 64Cu-labeled chelator-somatostatin conjugate is related to the dissociation of the radio-copper from its chelator. The 64Cu complex of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) has demonstrated instability in vivo, whereas 64Cu-CB-TE2A (CB-TE2A is 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane) was highly stable. METHODS: Receptor binding, nuclear uptake, internalization, and efflux assays were performed to characterize the interaction with the somatostatin receptor and the intracellular fate of 64Cu-labeled chelator-peptide conjugates in A427-7 cells. From these data, the absorbed dose to cells was calculated. RESULTS: 64Cu-TETA-Y3-TATE (64Cu-[1]) and 64Cu-CB-TE2A-Y3-TATE (64Cu-[2]) had high affinity for somatostatin receptor subtype 2 (SSTr2) in A427-7 cells. After 3 h, 64Cu-[2] showed greater internalization (>30%) compared with 64Cu-[1] (approximately 15%). There was uptake of 64Cu-[1] in nuclei of 427-7 cells (9.4% +/- 1.7% at 24 h), whereas 64Cu-[2] showed minimal nuclear accumulation out to 24 h (1.3% +/- 0.1%). A427-7 cells were exposed to 0.40 Gy from 64Cu-[1] and exposed to 1.06 Gy from 64Cu-[2]. External beam irradiation of A427-7 cells showed <20% cell killing at 1 Gy. CONCLUSION: These results are consistent with our hypothesis that dissociation of 64Cu from TETA leads to nuclear localization. Dosimetry calculations indicated that the nuclear localization of 64Cu-[1] was not significant enough to increase the absorbed dose to the nuclei of A427-7 cells. These studies show that 64Cu localization to cell nuclei from internalizing, receptor-targeted radiopharmaceuticals is related to chelate stability.

Characterization of somatostatin receptor subtype 2 expression in stably transfected A-427 human cancer cells.

Although radiolabeled somatostatin analogs have become highly prevalent in the diagnosis and treatment of somatostatin receptor subtype (sst)-positive tumors, there are relatively few options with respect to sst-positive tumor cell lines and animal models. It would be highly beneficial, particularly for therapeutic purposes, to have several clones of one human sst2-positive cell line that express a range of sst2 concentrations for evaluating the dose response and intracellular processing of radiolabeled somatostatin analogs. The human non-small cell lung cancer line A-427 was stably transfected with a hemagglutinin-tagged human sst2. Expression of the receptor was evaluated in vitro using flow cytometry, saturation binding analysis, internalization assays, and quantitative polymerase chain reaction. The receptor expression was also validated in an in vivo mouse model in biodistribution and micro-positron emission tomography (microPET) studies using the somatostatin analog octreotide (OC), which was linked to the (64)Cu chelator 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), or (64)Cu-TETA-OC. Stable clones were isolated, and four clones (2, 4, 5, and 7) were chosen for further examination. In vitro assays showed that clone 4 had no expression of sst2, whereas the others had various levels in the order of 7 > 2 > 5. Biodistribution studies with (64)Cu-TETA-OC showed the same rank order, with tumor uptake of the clones ranging from 0.8 to 6.5% injected dose/g. These studies showed that there was a strong correlation among the in vitro assays and between the in vitro assays and the biodistribution. MicroPET confirmed significant uptake of (64)Cu-TETA-OC in clone 7 and background uptake in clone 4. These studies show that clones of a human cell line can be produced expressing various levels of sst2 that should be useful for the future evaluation of radiolabeled somatostatin analogs.