Quantitative PET of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody
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- Cai, W., Chen, K., He, L. et al. Eur J Nucl Med Mol Imaging (2007) 34: 850. doi:10.1007/s00259-006-0361-6
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Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab.
We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), labeled with 64Cu, and tested the resulting 64Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated.
MicroPET imaging showed that 64Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R2 = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver.
The success of EGFR-positive tumor imaging using 64Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents.
KeywordsCetuximabEpidermal growth factor receptorMicro-positron emission tomographyCopper-64
Epidermal growth factor receptor (EGFR) is a member of the erbB family of tyrosine kinase receptors . It is a 170-kDa cell surface protein composed of three regions: an intracellular domain with adenosine triphosphatase (ATP)-dependent tyrosine kinase activity, a hydrophobic transmembrane domain, and an extracellular ligand binding domain . Many epithelial cancers over-express EGFR, and dysregulation of EGFR is associated with several key features of cancer such as autonomous cell growth, inhibition of apoptosis, angiogenic potential, invasion, and metastases [3, 4].
Many monoclonal antibody (mAb) products have been approved for clinical use in the United States, and dozens more are currently in clinical evaluation [5, 6]. Cetuximab (Erbitux; C255; ImClone, Somerville, NJ and Bristol-Myers Squibb, New York, NY), an immunoglobulin G1 mouse–human chimeric monoclonal antibody that binds with high affinity to EGFR, was the first anti-EGFR mAb to be evaluated clinically . By blocking the EGFR signal transduction pathway, cetuximab can cause G1 phase cell cycle arrest and disruption of cell cycle progression which occurs through inhibition of DNA repair mechanisms and survival pathways, decrease in angiogenesis and cellular adhesion, and inhibition of matrix metalloproteinases required for metastatic invasion . It was recently approved by FDA for the treatment of patients with EGFR-expressing metastatic colorectal cancer. Cetuximab (either alone or in combination with other treatment modalities) is also currently in advanced clinical trials in several other types of solid tumor, such as head and neck cancer , non-small cell lung cancer (NSCLC) , pancreatic cancer , cervical cancer, recurrent epithelial ovarian cancer, primary peritoneal carcinoma, and rectal cancer .
99mTc-labeled cetuximab has been reported for single-photon emission computed tomography (SPECT) imaging of EGFR expression . This tracer has reasonable dosimetric properties for a diagnostic nuclear medicine agent. The potential of the long-lived positron emitter 89Zr in predicting the biodistribution of therapeutic radiometals (such as 88Y, 90Y, and 177Lu) when labeled to cetuximab via different types of chelator has been evaluated . 68Ga-DOTA-hEGF has also been reported for EGFR imaging .
Since many cancer types over-express EGF and EGFR, detection of EGFR over-expression can provide important diagnostic information, which can influence patient management. For positron emission tomography (PET) imaging of EGFR expression, 64Cu-labeled mAb is of particular interest since the half-life of 64Cu (12.7 h) is suitable for the targeting and clearance kinetics of the mAb . Although cetuximab is currently in the clinic, no quantitative PET imaging using 64Cu-labeled cetuximab has been reported. Quantitative non-invasive measurement of EGFR expression level in vivo is critical in the patient selection for cetuximab treatment or clinical trials and monitoring of treatment response. Herein we describe the use of 64Cu-labeled cetuximab for PET imaging of EGFR expression in vivo, and the tracer uptake measured by PET scans was correlated with the EGFR expression measured by western blotting.
Materials and methods
All commercially available chemical reagents were used without further purification, 1,4,7,10-Tetraazadodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) was purchased from Macrocyclics, Inc. (Dallas, TX) and Chelex 100 resin (50–100 mesh) was purchased from Aldrich (St. Louis, MO). Water and all buffers were passed through Chelex 100 column (1 × 15 cm) before use in radiolabeling procedures to ensure that the aqueous buffer is heavy metal free. PD-10 desalting columns were purchased from GE Healthcare (Piscataway, NJ). Athymic nude mice were obtained from Harlan (Indianapolis, IN) at 4–5 weeks of age. 64Cu was provided by Washington University in St. Louis and University of Wisconsin, Madison.
Cell lines and xenografts
The U87MG human glioblastoma, PC-3 human prostate carcinoma, CT-26 murine colorectal carcinoma, HCT-8, HCT-116, and SW620 human colorectal carcinoma, and MDA-MB-435 human breast cancer cell lines were obtained from the American Type Culture Collection (ATCC) and maintained under standard conditions [17–19]. All animal experiments were performed under a protocol approved by the Stanford University Administrative Panel on Laboratory Animal Care (A-PLAC). All subcutaneous tumor models were established in 4- to 6-week-old female athymic nude mice except for the PC-3 tumor, where male mice were used. Typically 5 × 106 cells suspended in 100 μl serum-free corresponding culture medium (Invitrogen Inc., Carlsbad, CA) were injected into the front flank of the mice. MDA-MB-435 breast cancer model was established by orthotopic injection of 5 × 106 cells into the mammary fat pad of female athymic nude mice . The mice were used for microPET imaging studies when the tumor volume reached 200–400 mm3 (1–4 weeks after inoculation of the cancer cells).
DOTA conjugation and radiolabeling
Number of DOTA per cetuximab and immunoreactive fraction
MicroPET and image analysis
Details of the PET procedure have been reported earlier [26, 27]. Briefly, PET scans were performed using a microPET R4 rodent model scanner (Concorde Microsystems Inc., Knoxville, TN). Scanner calibration was performed to map between microPET image units and units of radioactivity concentration. Mice were injected with 5–10 MBq of 64Cu-DOTA-cetuximab via the tail vein under isoflurane anesthesia, and 2–20 min static PET scans were performed at various time points post injection (p.i.). The images were reconstructed by a two-dimensional ordered subsets expectation maximum algorithm and no correction was applied for attenuation or scatter . For each microPET scan, three-dimensional regions of interests (ROIs) were drawn over the tumor and major organs by using vendor software ASI Pro 22.214.171.124 on decay-corrected whole-body coronal images. Values in three to ten adjacent slices (depending on the size of the tumor or organ) were averaged to obtain a reproducible value of activity concentration in the ROI drawn. Assuming a tissue density of 1 g/ml, the ROIs were converted to MBq/g per minute using a conversion factor, and then divided by the administered activity to obtain an imaging ROI-derived percent injected dose per gram of tissue (%ID/g).
After the microPET studies and after the radioactivity had mostly decayed, the mice were sacrificed and the tumor tissues were harvested. Tumor tissue protein was extracted using T-PER tissue protein extraction buffer and the concentration was determined using microBCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL). After SDS-PAGE separation of 40 μg of total protein, it was transferred to a polyvinylidene fluoride membrane (Invitrogen Corp., Carlsbad, CA) and incubated at room temperature with 5% non-fat milk blocking buffer. The blots were then incubated overnight at 4°C with cetuximab, followed by incubation at room temperature for 1 h with HRP-conjugated anti-human antibody (GE Healthcare, Piscataway, NJ). The bands were detected using the ECL western blotting detection system (GE Healthcare). Tubulin was used as loading control. After development, films were scanned with a scanner using grayscale mode. The images were opened in ImageJ, a Java-based image processing software. An ROI was drawn over the first lane, and was applied for other lanes and the background to obtain densitometric signal from equal area. Background signal was subtracted before normalization. Three samples of each tumor model were prepared for western blot to obtain semiquantitative data for statistical analysis.
Two representative tumors (PC-3 and MDA-MB-435, with high and low tracer uptake, respectively) were chosen for qualitative evaluation of the tumor EGFR expression. Frozen tumor sections (5 μm thick) were warmed to room temperature, fixed with ice-cold acetone for 10 min, and then dried in the air for 30 min. The sections were rinsed in PBS for 2 min and blocked in 10% donkey serum for 1 h at room temperature. The sections were incubated with cetuximab (100 μg/ml) for 1 h at room temperature and visualized with FITC-conjugated donkey anti-human secondary antibody (1:200, Jackson ImmunoResearch Laboratories, Inc.) under a microscope (Carl Zeiss Axiovert 200M, Carl Zeiss USA, Thornwood, NY). Images were taken under the same condition and displayed at the same scale.
Radiation dosimetry extrapolation to human
Estimated human dosimetry was calculated from microPET imaging results on Sprague-Dawley female rats (Harlan) injected with about 37 MBq of 64Cu-DOTA-cetuximab. The rats were scanned at two bed positions in order to cover the whole body, and ROI analysis was carried out on major organs [22, 24, 29]. Time-activity curves were generated from the mean values obtained in rats for each organ of interest. We then calculated source organ residence times for the human model by integrating a monoexponential fit to the experimental biodistribution data for major organs and the whole body. The source organ residence times obtained forthwith were used with a standard quantitation platform Organ Level Internal Dose Assessment (OLINDA; Vanderbilt University) . Projected radiation absorbed doses for humans were made by assuming that, for the purpose of this exercise, the metabolism rates and pharmacokinetics of 64Cu-DOTA-cetuximab in man and mouse are equivalent. Therefore, the biodistribution data were assumed to apply to a reference adult human subject. The whole-body time-activity curve was integrated by fitting the whole-body retention data to a biphasic exponential decay curve, which represented the retention data more closely than a single exponential equation.
Quantitative data were expressed as mean±SD. Means were compared using one-way ANOVA and Student’s t test. P values <0.05 were considered statistically significant.
Radiolabeling yield, specific activity, number of DOTA per cetuximab, and immunoreactive fraction
The radiolabeling procedure took about 85 ± 10 min (n = 5) and the radiochemical yield was 84.0 ± 8.7% (based on 74 MBq of 64Cu per 50 μg of DOTA-cetuximab). The specific activity of 64Cu-DOTA-cetuximab was 1.24 ± 0.13 GBq/mg cetuximab. 64Cu-DOTA-cetuximab was determined to have 21.5 ± 2.2 DOTA residues per cetuximab (n = 4). Using U87MG cells, the immunoreactive fraction was measured to be 62.6 ± 13.7%, indicating that the accessible lysine residues were largely located away from the complementarity-determining region.
MicroPET of tumor models with different EGFR expression
Correlation of 64Cu-DOTA-cetuximab uptake with EGFR expression level
Human dosimetry extrapolation
Estimated radiation absorbed doses to an adult human after intravenous injection of 64Cu-DOTA-cetuximab based on the microPET imaging data obtained in Sprague-Dawley rats (n = 3)
Although cetuximab is already in the clinic and multiple advanced stage clinical trials [9–12], there has been no quantitative PET imaging of EGFR expression reported using radiolabeled cetuximab. In this study we successfully demonstrated that 64Cu-labeled cetuximab exhibits high receptor specificity both in vitro and in vivo. Most importantly, there is a good linear correlation (R2 = 0.80) between the tumor uptake (measured by PET scans) and the EGFR expression level in vivo (measured by western blotting).
Non-invasive molecular imaging can give whole-body readout in live animals; help to decrease the workload and number of animals used; provide more statistically relevant results than in vitro studies as longitudinal studies can be performed in the same animal which can serve as its own control; aid in lesion detection in cancer patients and patient stratification; and help in individualized anticancer treatment monitoring and dose optimization . Through the development of a robust imaging platform, molecular imaging can dramatically facilitate and speed up many steps of anticancer drug development in both the preclinical and clinical stages. The future of cancer therapy, molecular/personalized medicine, can only be achieved through quantitative molecular imaging. PET is a quantitative imaging modality and the agreement between the tumor/organ uptake measured by invasive biodistribution studies and the non-invasive PET scans has been vigorously validated [22, 24, 33]. Quantitative PET imaging of molecular cancer marker, such as EGFR, expression in vivo is critical in early and sensitive lesion detection, patient selection for entering clinical trials, better treatment monitoring and dose optimization based on non-invasive detection of early response to anticancer treatment, and elucidation of the mechanisms of treatment efficacy underlying the relevant signaling pathways.
Owing to the relatively high molecular weight of the mAb (150 kDa), 64Cu-DOTA-cetuximab clears from the body mainly through the hepatic route. The serum half-lives of 64Cu-DOTA-cetuximab in both mice and rats are less than 18 h based on microPET studies, much shorter than that in human , presumably because of the much faster metabolic rate of murine species as compared with human. Dosimetry of 99mTc-labeled cetuximab has been reported in cancer patients . Although mAb-based tracer mainly excretes through the hepatic pathway, the highest radiation dose of 99mTc-cetuximab is to the kidney rather than the liver, indicating low stability of the 99mTc-labeled immunoconjugate. In this study, no kidney uptake was observed even at late time points (48 h), indicating that the 64Cu-DOTA complex is quite stable in vivo. The OLINDA software, as a replacement for MIRDOSE3, provides a calculation of the effective dose for human adults as defined in ICRP publication 60 . The use of rodent biodistribution data is generally thought to give a worst-scenario estimate of absorbed doses to normal organs; whether this is true for 64Cu-DOTA-cetuximab remains to be tested in human patients. Nevertheless, for imaging purposes, the administered radiation dose is unlikely to cause any adverse effects since the amount injected is usually quite low (about 400 MBq) .
The half-life of 64Cu is 12.7 h. In this study we followed the tracer uptake for 48 h (about four half-lives of 64Cu). Longer term monitoring may be necessary for future studies and 89Zr (t1/2 79.3 h) or 124I (t1/2 100.8 h) may be used. It has been reported that 89Zr-labeled cetuximab reflects the biodistribution of 90Y-labeled cetuximab . The 1:1 chelator/mAb ratio gave a significantly higher immunoreactive fraction (about 95%) of the resulting 89Zr- or 90Y-labeled cetuximab because of less disturbance on the antigen binding domain compared with the about 20 DOTA residues per cetuximab in our study. However, the tracer uptake in the tumor was comparable (15–25%ID/g). Although reducing the number of DOTA per cetuximab may increase the radioimmunoreactive fraction, the specific activity of 64Cu-DOTA-cetuximab will also be lower, which will lead to lower tumor uptake values . There is a fine balance between the immunoreactive fraction and specific activity/tumor uptake and this may require more investigation in future studies.
In reality, it may be difficult to determine whether a tumor is EGFR positive if the tracer uptake in the tumor is not very high. The enhanced permeability and retention (EPR) effect (due to the leaky vasculature and lack of lymphatic drain in the tumor) may cause a certain level of tracer uptake in the tumor . Tumor uptake solely attributed to the EPR effect has a characteristic pattern of reaching a maximum at early time points (the actual time differs between different tumors) followed by steady decrease afterwards. For specific targeting attributed to mAb–antigen interaction, the tumor uptake increases rapidly over time and reaches a plateau and remains steady, such as the U87MG and PC-3 tumor uptake observed in this study (Fig. 3b). Clinical translation of 64Cu-DOTA-cetuximab will be critical for the maximum benefit of cetuximab-based anticancer agents as imaging provides a straightforward and convenient way to monitor the biological changes at the molecular level in vivo. Multiple time point PET scans of the same patient over time may be required, and analysis of the tumor uptake pattern is suggested in order to differentiate EGFR specific activity accumulation from passive targeting due to the EPR effect.
We describe here the quantitative PET imaging of EGFR expression using 64Cu-labeled cetuximab, a chimeric mAb against EGFR. MicroPET imaging of 64Cu-DOTA-cetuximab showed prominent uptake in EGFR-expressing tumors but low accumulation in EGFR-negative tumors. Most importantly, there was a good linear correlation between the %ID/g values (measured by PET scans) and the EGFR expression level (measured by western blotting). The success of EGFR-specific tumor imaging using 64Cu-DOTA-cetuximab may be translated into the clinic. It may significantly expedite the process of clinical evaluation of cetuximab and provide maximum benefit in treating various types of EGFR-expressing cancer.
This project was financially supported by National Institute of Biomedical Imaging and Bioengineering (NIBIB) (R21 EB001785), National Cancer Institute (NCI) (R21 CA102123, P50 CA114747, U54 CA119367, and R24 CA93862), Department of Defense (DOD) (W81XWH-04-1-0697, W81XWH-06-1-0665, W81XWH-06-1-0042, and DAMD17-03-1-0143), and a Benedict Cassen Postdoctoral Fellowship from the Education and Research Foundation of the Society of Nuclear Medicine (to W.Cai).