Small-Animal PET Imaging of Human Epidermal Growth Factor

Bioconjugate Chem. 2010, 21, 947–954

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Small-Animal PET Imaging of Human Epidermal Growth Factor Receptor Positive Tumor with a 64Cu Labeled Affibody Protein Zheng Miao,†,‡,§ Gang Ren,†,‡,§ Hongguang Liu,†,‡,§ Lei Jiang,†,‡,§ and Zhen Cheng*,†,‡,§ Molecular Imaging Program at Stanford (MIPS), Department of Radiology, and Bio-X Program, Stanford University, Stanford, California 94305-5344. Received November 20, 2009; Revised Manuscript Received February 25, 2010

Epidermal growth factor receptor (EGFR) has become an attractive target for cancer molecular imaging and therapy. Affibody proteins against EGFR have been reported, and thus, we were interested in evaluating their potential for positron emission tomography (PET) imaging of EGFR positive cancer. An Affibody analogue (AcCys-ZEGFR:1907) binding to EGFR was made through conventional solid phase peptide synthesis. The purified protein was site-specifically coupled with the 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidethylacetamide (maleimido-mono-amide-DOTA) to produce the bioconjugate, DOTA-ZEGFR:1907. 64Cu labeled probe 64Cu-DOTA-ZEGFR:1907 displayed a moderate specific activity (5-8 MBq/nmol, 22-35 µCi/µg). Cell uptake assays by pre-incubating without or with 300 times excess unlabeled Ac-Cys-ZEGFR:1907 showed high EGFRspecific uptake (20% applied activity at 0.5 h) in A431 epidermoid carcinoma cancer cells. The affinity (KD) of 64 Cu-DOTA-ZEGFR:1907 as tested by cell saturation analysis was 20 nM. The serum stability test showed excellent stability of the probe with >95% intact after 4 h of incubation in mouse serum. In vivo small-animal PET imaging showed fast tumor targeting, high tumor accumulation (∼10% ID/g at 1 h p.i.), and good tumor-to-normal tissue contrast of 64Cu-DOTA-ZEGFR:1907 spiked with a wide dose range of Ac-Cys-ZEGFR:1907. Bio-distribution studies further demonstrated that the probe had high tumor, blood, liver, and kidney uptakes, while blood radioactivity concentration dropped dramatically at increased spiking doses. Co-injection of the probe with 500 µg of AcCys-ZEGFR:1907 for blocking significantly reduced the tumor uptake. Thus, 64Cu-DOTA-ZEGFR:1907 showed potential as a high tumor contrast EGFR PET imaging reagent. The probe spiked with 50 µg of Ac-Cys-ZEGFR:1907 improved tumor imaging contrast which may have important clinical applications.

INTRODUCTION Epidermal growth factor receptor 1 (EGFR) is a transmembrane protein belonging to the ErbB receptor kinase family. Overexpression of EGFR has been frequently detected in a wide range of human tumors, for example, small cell lung cancer, small cell carcinoma of the head and neck, esophageal cancer, gastric cancer, gliomas, colon cancer, pancreas cancer, breast cancer, ovarian cancer, bladder cancer, kidney cancer, prostate cancer, etc. (1, 2). Cetuximab, Lapatinib, Gefitinib, and Erlotinib have shown clinical benefits in lung, colorectal, pancreatic, and breast cancer patients by targeting over-expressed/activated EGFR (3-5). Furthermore, increasing evidence has demonstrated that there is a correlation between EGFR overexpression and tumor metastasis formation, therapy resistance, poor prognosis, and short survival for some cancer types. It has been found that EGFR expression level is a strong prognostic indicator for head and neck, ovarian, cervical, bladder, and esophageal cancer (6, 7). Therefore, EGFR has become an attractive target for cancer molecular imaging and therapy. A variety of small molecules based upon EGFR substrates and EGF have been labeled with different radionuclides for molecular imaging of EGFR expression and activity. However, the small molecules and EGF based probes generally show rapid * To whom correspondence should be addressed. Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, 1201 Welch Road, Lucas Expansion, P020A, Stanford University Stanford, CA 94305. Tel: 650-723-7866. Fax: 650-736-7925. E-mail: [email protected]. † MIPS. ‡ Department of Radiology. § Bio-X Program.

blood clearance and very low tumor uptake; thus, the imaging quality is poor (8-10). Radiolabeled monoclonal antibodies (MAbs) against EGFR demonstrate good tumor uptake, but the tumors can only be visualized at several hours or even days after the injection of probes because of both slow tumor targeting ability and slow clearance of the radiolabeled MAbs (11, 12). Recently, small protein scaffolds have shown great potential for recognizing a variety of biomarkers (13-17). Among them, Affibody molecules have demonstrated to be a promising universal platform for developing imaging or therapeutic agents for different molecular targets (17-19). Affibody molecules are small, engineered proteins with 58-amino acid residues and a three-helix bundle scaffold structure, yet at the same time they display a binding surface as large as that of antibodies. In our previous research, Affibody ZHER2:477 site-specifically labeled with 18F and 64Cu has shown rapid human epidermal growth factor receptor 2 (HER2) positive tumor targeting ability and good tumor-to-normal tissue imaging contrast at even 1 h postinjection (p.i.) of the probe (20, 21). These results have encouraged us to further explore this type of protein scaffold for positron emission tomography (PET2) imaging of many other important tumor biomarkers including EGFR. Several anti-EGFR Affibody proteins (ZEGFR) with high affinities have been reported recently (22, 23). Some of them were radiolabeled with 111In in a nonsite-specific manner and evaluated using EGFR-expressing A431 tumor xenografts. Biodistribution studies demonstrated that these Affibody molecules displayed specific and good tumor accumulation; especially, ZEGFR:1907 was reported to show the best in vivo tumor 2 Abbreviations: DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid; PET, positron emission tomography; HPLC, highperformance liquid chromatography; p.i., postinjection.

10.1021/bc900515p  2010 American Chemical Society Published on Web 04/19/2010

948 Bioconjugate Chem., Vol. 21, No. 5, 2010

Figure 1. Anti-EGFR Affibody protein and

Miao et al.

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Cu radiolabeling of DOTA-ZEGFR:1907 for EGFR imaging.

targeting properties. In this study, we aimed to develop an Affibody based PET probe for the imaging of an EGFR positive tumor. A ZEGFR:1907 analogue, Ac-Cys-ZEGFR:1907, was first synthesized so that the protein can be site-specifically conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10maleimidethylacetamide (Malei mido-mono-amide-DOTA) through the cysteine residue (Figure 1). The resulting bioconjugate, Ac-Cys(DOTA)-ZEGFR:1907 (abbreviated as DOTAZEGFR:1907), was then radiolabeled with a positron emitter 64Cu (half-life t1/2 ) 12.7 h, Eβ+max ) 656 keV, and β+ decay ) 19%) because it can be produced in large quantities and high specific activity. The ability of 64Cu-DOTA-ZEGFR:1907 to image the EGFR positive tumor using small-animal-PET was then evaluated on the epidermoid carcinoma A431 tumor xenograft mice model.

MATERIALS AND METHODS General. Maleimido-mono-amide-DOTA was purchased from Macrocyclics Inc. Dimethylsulfoxide (DMSO) and ethyl acetate were purchased from Fisher Scientific. Dichloromethane (DCM), trifluoroacetic acid (TFA), thioanisole (TIS), ethanedithiol (EDT), ethylene-diamine-tetra-acetic acid (EDTA), triethylamine, octanol, N-hydroxybenzotriazole hydrate (HOBT), 1-ethyl3-[3-dimethylaminopropyl]carbodiimide (EDC), N,N-diisopropylethylamine (DIEA), ethyl acetate, dithiothriotol (DTT), and all other standard synthesis reagents were purchased from Sigma-Aldrich Chemical Co. The fluorescent dye 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) was purchased from Molecular Probes Inc. All chemicals were used without further purification. The radionuclide, 64Cu, was provided by the Department of Medical Physics, University of Wisconsin at Madison. Human epidermoid carcinoma cancer cell line A431 was obtained from the American Type Tissue Culture Collection. Analytical RP-HPLC columns (Dionex, Acclaim-120 C18, 4.6 mm ×250 mm) with a guard column were used for analysis of 64Cu labeled protein and unlabeled protein. The mobile phase was solvent A, 0.1% trifluoroacetic acid (TFA)/H2O, and solvent B, 0.1%TFA/acetonitrile. The flow rate was 1 mL/min, with the mobile phase starting from 95% solvent A and 5% solvent B (0-3 min) to 35% solvent A and 65% solvent B at 33 min, then going to 15% solvent A and 85% solvent B (33-36 min), maintaining this solvent composition for another 3 min (36-39 min) and returning to initial solvent composition by 42 min. All other general materials (e.g., mice) and instruments (e.g., radioactive dose calibrator and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), liquid chromatography electrospray ionization mass spectrometer (LC-ESI-MS), etc.) were the same as those previously reported (20). Conjugation of DOTA-ZEGFR:1907. Ac-Cys-ZEGFR:1907 (AcCVDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIA SLVDDPSQSANLLAEAKKLNDAQAPK-NH2) was synthesized on

an automatic peptide synthesizer (CS Bio, CS 336X). Briefly, Rink Amide LS resin (200 mg, 40 µmol, Advanced ChemTech, 0.2 mmol/g loading) was swollen in N,N-dimethylformamide (DMF) for 30 min. Fmoc-protected amino acids were purchased from Novabiochem/EMD Chemicals Inc. Fmoc groups were removed with 20% piperidine in DMF. The aliquots of amino acids (0.20 mmol) were activated in a solution containing 0.20 mmol of HOBt and 0.5 M diisopropylcarbodiimide (DIC) in DMF. The small protein was N-terminal-acetylated by activation of acetic anhydride with HOBt/DIEA after deprotection of the Fmoc protection group of the last amino acid residue. Peptide cleavage and deprotection were carried out by a 3-h incubation in a mixture of TFA/TIS/EDT/H2O (94:2:2:2). The mixture was filtered, and the peptide in solution was precipitated with anhydrous diethyl ether. The resulting peptide was washed four times with ice-cold anhydrous diethyl ether, dried, and dissolved in 1 mM DTT. The peptide was purified by RP-HPLC on a C-18 column. Fractions were collected and lyophilized. The target product was characterized by ESI-MS and was ready for use in the next step in the reaction. The general procedure for the conjugation of maleimidomono-amide-DOTA with Ac-Cys-ZEGFR:1907 is as follows: AcCys-ZEGFR:1907 was dissolved with freshly degassed phosphate buffer (pH 7.4) at a concentration of 1 mg/mL. The bifunctional chelator maleimido-mono-amide-DOTA dissolved in DMSO (10 mM) was added at 20 equivalents per equivalent of the Affibody. After mixing by vortexing for 2 h, we purified the reaction mixture by a Dionex HPLC with a protein-and-peptide C4 column (Grace Vydac 214TP54). Characterization of the products was confirmed using MALDI-TOF-MS or LC-ESIMS. The purity of the bioconjugate DOTA-ZEGFR:1907 was confirmed by HPLC. The concentration of free cysteine side chains was determined with the fluorescent reagent CPM on both unconjugated and conjugated proteins. The method of Greiner et al. (24) was slightly modified as follows: β-mercaptoethanol standard solutions were prepared in MOPS buffer (1 mM MOPS, pH 7.9, 120 mM NaCl, 10 mM MgCl2, and 1.0 mM EDTA) at concentrations of 30, 20, 10, 5, 3, 1, 0.3, and 0.1 µM. The protein concentration was determined by the microBradford assay. A 15 µL aliquot of 0.4 mM CPM in DMF was added to 15 µL of each standard and each protein sample. After a 1-h incubation time at room temperature, triton X-100 (3 mL, 1%) was added to each reaction. The intensity of fluorescence emission was measured on 50 µL of samples on a NUNC plate, using a TECAN SAFIRE fluorescence plate reader. The excitation and emission wavelengths were 390 and 473 nm, respectively. 64 Cu Radiolabeling of DOTA-ZEGFR:1907. The Affibody conjugate DOTA-ZEGFR:1907 was radiolabeled with 64Cu by the addition of 66.6-148 MBq (2-4 mCi) 64CuCl2 (1 µg of DOTAZEGFR:1907 per 2.96 MBq 64Cu) in 0.1 N sodium acetate (NaOAc, pH 6.3) buffer followed by a 1-h incubation at 45 °C. EDTA

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Cu-Affibody PET Probe for EGFR Imaging

(5 µL, 10 mM) was then added to quench the free 64Cu. The radiolabeled complex was purified by a PD-10 column (GE Healthcare). The product was washed out by phosphate-buffered saline (PBS, pH 7.4) and passed through a 0.22-µm Millipore filter into a sterile vial for in vitro and animal experiments. Radioanalytical HPLC was used to analyze the purified 64CuDOTA-ZEGFR:1907. Octanol/Water Partition Coefficient. To determine the lipophilicity of 64Cu-DOTA-ZEGFR:1907, approximately 370 kBq of the probe in 500 µL of PBS (0.01 M, pH 7.4) was added to 500 µL of octanol in an Eppendorf microcentrifuge tube. The resulting biphasic system was mixed vigorously for 0.5 h and left at room temperature for another 0.5 h. The two phases were then separated by centrifugation at 3000g for 5 min (model 5415R Eppendorf microcentrifuge; Brinkman). From each layer, an aliquot of 100 µL was removed and counted in a γ-counter (Packard Instrument). The partition coefficient (log P) was then calculated as a ratio of counts in the octanol fraction to the counts in the water fraction. The experiment was repeated 3 times. Serum Stability. 64Cu-DOTA-ZEGFR:1907 (6.7 MBq, 180 µCi) in 250 µL of PBS was added to 1 mL of mouse serum (Sigma). After incubation at 37 °C for 1 h, 4 h, and 23 h, 74 kBq-118 kBq (20-40 µCi) solution was precipitated with 300 µL of ethanol. After centrifugation, the supernatant was transferred to a new Eppendorf tube, and DMF (300 µL) was added to precipitate the residue of serum protein. After centrifugation, the supernatant was acidified with 300 µL of buffer A (water + 0.1% TFA) and filtered through a Spin-X centrifuge tube filter (0.22 µm nylon, COSTAR). The filtrate was then injected into the radio-HPLC under conditions identical to the ones used for analyzing the original 64Cu-DOTA-ZEGFR:1907. In Vitro Cell Assays. The EGFR positive A431 cell line was cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen Life Technologies). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C, with the medium changed every two days. A 70-80% confluent monolayer was detached by 0.1% trypsin and dissociated into a single cell suspension for further cell culture. The in vitro cell uptake assays of the 64Cu-DOTA-ZEGFR:1907 were performed as previously described with minor modifications (23, 25). Briefly, the A431 cells were washed three times with PBS and dissociated with 0.25% trypsin-EDTA. DMEM medium was then added to neutralize trypsin-EDTA. Cells were spun down and resuspended with serum free DMEM. 5 × 105 cells were incubated at 37 °C for 0.25 to 2 h with the radioactive probe (7.4 kBq, ∼7 ng of Affibody) in 0.5 mL of serum-free DMEM medium. The nonspecific binding of the probes with A431 cells was determined by coincubation with nonradiolabeled DOTA-ZEGFR:1907 (final concentration 0.6 µM). The cells were washed three times with 0.01 M PBS (pH 7.4). Cells were then washed three times with chilled PBS and spun down at a speed of 7000-8000 rpm. The cell pellets at the bottom of the tube were spliced, and the radioactivity of the pellets was measured using a γ-counter (PerkinElmer 1470, Waltham, MA). The uptake (counts/min) was normalized to the percentage of binding for analysis using Excel (Microsoft). The receptor saturation assay of 64Cu-DOTA-ZEGFR:1907 was conducted with the A431 cells, and 3 × 105 cells were plated on 6-well plates one day before the experiment. Cells were washed with PBS three times. Serum free DMEM (1 mL) was added to each well, and 8.9 kBq-532.8 kBq (0.24 µCi -14.4 µCi, 2 nM-120nM final concentration) of 64Cu-DOTAZEGFR:1907 with or without 100 times excess of Ac-CysZEGFR:1907 was added to the plates. The plates were incubated on ice for 2 h. Then cells were washed with cold PBS three

Bioconjugate Chem., Vol. 21, No. 5, 2010 949

times and detached with TrypLE-Express (Invitrogen, San Diego, CA). The radioactivity of the cells was measured in a γ-counter. The data was analyzed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA), and the KD value of DOTA-ZEGFR:1907 was calculated from a one-site fit binding curve. Biodistribution Studies. All animal studies were carried out in compliance with federal and local institutional rules for the conduct of animal experimentation. The A431 cells (5 × 106) suspended in PBS were implanted subcutaneously in the right upper shoulders of nude mice. Tumors were allowed to grow to around 0.4-0.6 cm in diameter (1-2 weeks). The tumor bearing mice were subject to in vivo biodistribution and imaging studies. For biodistribution studies, the A431 tumor-bearing mice (n ) 3 for each group) were injected with 64Cu-DOTA-ZEGFR:1907 (1.11-1.85 MBq, 30-50 µCi, 1-2 µg) spiked with different doses of nonradioactive Ac-Cys-ZEGFR:1907 (0, 5, 50, and 500 µg) through the tail vein and sacrificed at different time points (1, 4, and 24 h) p.i. Tumor and normal tissues of interest were removed and weighed, and their radioactivity was measured in a γ-counter. The radioactivity uptake in the tumor and normal tissues was expressed as a percentage of the injected radioactivity (decay corrected) per gram of tissue (% ID/g). Small-Animal PET Imaging. PET imaging of tumor-bearing mice was performed on a microPET R4 rodent model scanner (Siemens Medical Solutions USA, Inc.). The mice bearing A431 were injected with 64Cu-DOTA-ZEGFR:1907 (1.11-1.85 MBq, 30-50 µCi, 1-2 µg) spiked with cold Ac-Cys-ZEGFR:1907 (0, 5, 50, and 500 µg) via the tail vein. At different times after the injection, the mice were anesthetized with 2% isoflurane and placed in the prone position and near the center of the field of view of a microPET. The 3-min static scans were obtained, and the images were reconstructed by a two-dimensional ordered subsets expectation maximum (OSEM) algorithm. No background correction was performed. The method for quantification analysis of the images was the same as that reported previously (20). EGFR Western Analysis of Tumor Cells and Tissues. Tumor, liver, and kidneys from A431 xenografted mice were harvested and immediately frozen in dry ice. Thawed tissues were sliced into pieces. RIPA cell lysis buffer (Sigma) supplemented with protease inhibitor cocktail was added to the sliced tissue samples and incubated for 10 min in ice. The supernatant was collected by centrifuging at 14,000 rpm for 10 min at 4 °C. The protein concentrations of the samples were measured using the Bradford assay (BioRad, Hercules, CA). An equal amount of protein from each sample was loaded onto a 10% NuPAGE Bis-Tris gel and electroblotted to a PVDF membrane. After blocking with Tris buffered saline (TBS) plus 0.05% Tween 20 containing 5% powdered milk, the membrane was divided into two pieces at the 60 kDa marker. The top and bottom membranes were incubated separately with a rabbit monoclonal anti-human EGFR antibody (Cell Signal Technology) (1:1000) or mouse monoclonal anti-actin (Sigma-Aldrich) (1:1000) overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen) (1:1,000) or HRP-conjugated goat anti-mouse IgG (1:1,000), respectively for 1 h. After extensive washing, the protein bands were visualized using ECL plus (Invitrogen). Statistical Methods. Statistical analysis was performed using Student’s two-tailed t-test for unpaired data. A 95% confidence level was chosen to determine the significance between groups, with P < 0.05 being significantly different.

RESULTS Chemistry and Radiochemistry. Ac-Cys-ZEGFR:1907 with a cysteine residue at the N-terminal was successfully synthesized


Miao et al.

Figure 2. HPLC radiochromatograms of purified 64Cu-DOTA-ZEGFR:1907 (A) and radiolabeled probe after 1 h (B), 4 h (C), and 23 h (D) of incubation with mouse serum.

Figure 3. Characterization of EGFR-specific binding. (A) Cell uptakes of 64Cu-DOTA- ZEGFR:1907 in A431 cells over time at 37 °C with or without the presence of nonradioactive Affibody molecules DOTA-ZEGFR:1907. All results are expressed as the mean of triplicate measurements ( standard deviation. (B) Saturation assay using SKOV3 cells plotted by concentration of total radioligands versus bound radioligand. NSB, nonspecific binding; TB, total binding; and SB, specific binding.

through conventional solid phase peptide synthesis and purified by semi-preparative HPLC. The purified peptide was generally obtained in about 10% yield. The retention time on analytical HPLC for Ac-Cys-ZEGFR:1907 was found to be 26 min. The purified Ac-Cys-ZEGFR:1907 was characterized by MALDI-TOFMS. The measured molecular weight (MW) was consistent with the expected MW: m/z ) 6690.0 for [M + H]+ (calculated MW[M+H]+ ) 6689.6). Ac-Cys-ZEGFR:1907 was then conjugated with maleimido-mono-amide-DOTA and purified by C4 analytical HPLC. Analysis of free thiol of the DOTA-ZEGFR:1907 confirmed the absence of unreacted Ac-Cys-ZEGFR:1907 (data not shown). Furthermore, mass spectrometry analysis of the final product also only showed the expected mass peak for DOTAZEGFR:1907. The measured MW was m/z ) 7215 for [M + H]+ (calculated MW[M+H]+ ) 7215.1). The recovery yield was 47% after purification, and the purity for the final product was over 95% (retention time: 26 min). DOTA-ZEGFR:1907 was successfully labeled with 64Cu at 45 °C for a 1-h incubation (Figure 1). The labeling yield with 64Cu

was generally over 50%. The purification of radiolabeling solution using a PD-10 column afforded 64Cu-DOTA-ZEGFR:1907 with >95% radiochemical purity (Figure 2A, retention time )26 min) with modest specific activity (5-8 MBq/nmol, 22-35 µCi/ µg) at the end of synthesis (EOS). From the octanol-water partition coefficient measurements, the log P value of 64CuDOTA- ZEGFR:1907 was determined to be -1.01 ( 0.09, indicating a moderate hydrophilicity of the radiolabeled Affibody. In Vitro Assay of 64Cu-DOTA-ZEGFR:1907. The serum stability study showed that 64Cu-DOTA-ZEGFR:1907 had excellent resistance to proteolysis and transchelation with >95% of the probe intact after a 4-h incubation in the serum, and there was ∼85% intact 64Cu-DOTA-ZEGFR:1907 after 24 h of incubation (Figure 2). The receptor-binding affinity study of 64Cu -DOTAZEGFR:1907 for EGFR was performed using A431 cells (Figure 3B). The KD value of 64Cu-DOTA-ZEGFR:1907 was determined to be 20 nM from the saturation assay. Figure 3A shows the cell uptake of 64Cu-DOTA- ZEGFR:1907 in A431 cells at 37 °C over a 2-h incubation period. It was observed that the radiola-

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Table 1. Biodistribution Results for Carcinoma Cancera


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Cu-DOTA-ZEGFR:1907 in Nude Mice Bearing Subcutaneously Xenotransplanted A431 Human Epidermoid

1h

4h

organ (% ID/g) (spiked dose)

5 µg

0 µg

5 µg

tumor blood heart liver lungs muscle kidney spleen brain intestine skin stomach pancreas bone

9.72 ( 4.89 10.03 ( 3.80b 2.55 ( 0.77 22.81 ( 5.70 5.54 ( 0.45 0.59 ( 0.12 86.86 ( 25.59 2.57 ( 0.59 0.34 ( 0.15 3.28 ( 0.74 1.49 ( 0.44 3.85 ( 0.91 2.15 ( 1.22 1.54 ( 0.10

11.71 ( 1.98 11.68 ( 1.39c 3.35 ( 0.43c 18.99 ( 2.88 5.81 ( 0.36c 0.74 ( 0.06c 88.45 ( 14.72 3.09 ( 0.81 0.31 ( 0.06 3.49 ( 1.18 1.56 ( 0.21 3.87 ( 0.66 1.72 ( 0.38 1.08 ( 0.05

11.02 ( 1.41 4.84 ( 0.26c,d,f 1.97 ( 0.09c,d,f 20.03 ( 3.03d,f 4.02 ( 0.65c,f 0.53 ( 0.08c,d 126.51 ( 35.09 1.86 ( 0.26 0.22 ( 0.07 3.26 ( 0.22f 2.86 ( 0.83f 3.33 ( 0.43 1.95 ( 0.05d,f 1.32 ( 0.54f

T/blood T/muscle

1.23 ( 1.07b 16.29 ( 6.62

1.04 ( 0.01c 17.82 ( 3.66

2.29 ( 0.43c,f 20.98 ( 2.99

24 h 50 µg

f

500 µg (block)

5 µg

17.35 ( 4.48 1.81 ( 0.44d 1.50 ( 0.14d,e 13.27 ( 2.02d,e 3.07 ( 0.63 0.35 ( 0.06d 207.35 ( 101.16 2.02 ( 0.30 0.14 ( 0.07 3.65 ( 0.41e 2.08 ( 0.09e 2.73 ( 0.50 1.36 ( 0.21d,e 1.28 ( 0.54

6.15 ( 0.49 1.15 ( 0.05f 0.83 ( 0.08e,f 6.47 ( 1.52e,f 2.38 ( 0.13f 0.31 ( 0.11 151.41 ( 9.94 1.41 ( 0.26 0.12 ( 0.04 1.67 ( 0.12e,f 0.76 ( 0.08e,f 2.74 ( 0.33 0.60 ( 0.05e,f 0.43 ( 0.06f

10.46 ( 2.11 1.99 ( 0.59b 2.65 ( 0.48 12.83 ( 3.27 5.78 ( 2.28 0.74 ( 0.18 61.33 ( 18.67 2.33 ( 0.74 0.35 ( 0.09 4.43 ( 0.91 2.48 ( 1.05 3.88 ( 0.90 2.16 ( 0.72 1.33 ( 0.33

9.87 ( 2.99 49.46 ( 11.43e

5.84 ( 0.84f 20.98 ( 2.99e

5.39 ( 0.69b 16.05 ( 4.00

e

e,f

Uptake Ratios

a Data are expressed as the percentage of administered activity (injected probe) per gram of tissue (% ID/g) after intravenous injection of the probe (1.11-1.85 MBq, 30-50 µCi, 1-2 µg) spiked with 0, 5, 50, and 500 µg of Ac-Cys-ZEGFR:1907 at 1, 4, or 24 h (n ) 3). 64Cu-DOTA-ZEGFR:1907 demonstrates significantly higher uptake in A431 tumor mice than that of the blocked group (500 µg) at 4 h p.i. (P < 0.05). Student’s unpaired two tailed t-test was conducted. P < 0.05 was considered significant. b P < 0.05, comparing 5 µg of spike tracer biodistribution 1 h and 24 h p.i. c P < 0.05, comparing 0 µg and 5 µg of spike tracer biodistribution at 4 h p.i. d P < 0.05, comparing 5 µg and 50 µg of spike tracer biodistribution at 4 h p.i. e P < 0.05, comparing 50 µg spike and 500 µg (block) of dose tracer biodistribution at 4 h p.i. f >P < 0.05, comparing 5 µg spike and 500 µg (block) of dose tracer biodistribution at 4 h p.i.

beled peptide steadily increased over the 2-h incubation time, and it reached an EGFR specific uptake of ∼20% applied activity in 0.5 h. Pre-incubation with 300 time excess of DOTAZEGFR:1907 significantly blocked 64Cu-DOTA-ZEGFR:1907 uptake (P < 0.05 at all time points), demonstrating the receptor binding specificity of the PET probe. In Vivo Biodistribution Studies. The in vivo biodistribution studies of 64Cu-DOTA-ZEGFR:1907 were examined in a A431 xenograft mouse model, and the biodistribution data and statistical analysis results are presented in Table 1. For the probe spiked with 5 µg of Ac-Cys-ZEGFR:1907, high activity accumulation in the tumor was observed at early time points, with 9.72 ( 4.89 and 11.02 ( 1.41% ID/g uptake at 1 and 4 h p.i., respectively. 64Cu-DOTA-ZEGFR:1907 also showed good tumor retention with uptake of 10.46 ( 2.11% ID/g at 24 h. Blood radioactivity concentration was high at early time with 10.03 ( 3.80% ID/g at 1 h and dropped to 4.84 ( 0.26% ID/g at 4 h and 1.99 ( 0.59% ID/g at 24 h. High liver uptake and retention were also found (20.03 ( 3.03 and 12.83 ( 3.27%ID/g at 4 and 24 h p.i., respectively). At later time points such as 24 h p.i., 64Cu-DOTA-ZEGFR:1907 displayed a good tumor to blood ratio of 5.39 ( 0.69 (Table 1, 5 µg spiking dose). Excellent tumor-to-muscle ratios (>16) were found for all the time points studied. To study the spiking dose-dependent biodistribution of 64CuDOTA-ZEGFR:1907, 64Cu-DOTA-ZEGFR:1907 (1.11-1.85 MBq) was co-injected with 0, 5, and 50 µg of Ac-Cys-ZEGFR:1907 into A431 tumor bearing mice (Table 1). Tumor uptakes were maintained over 10% ID/g at 4 h p.i. for all different spiking doses. However, the blood radioactivity concentration of 64CuDOTA-ZEGFR:1907 was very different with uptake values of 11.68 ( 1.39, 4.84 ( 0.26, and 1.81 ( 0.44% ID/g at 0, 5, and 50 µg spiking dose respectively (4 h p.i., P < 0.05). Thus, high specific activity of 64Cu-DOTA-ZEGFR:1907 could not increase the tumor uptake, and the tumor-to-blood ratios at 0 and 5 µg were low (Table 1). Liver uptakes were also relatively high (18.99 ( 2.88 and 20.03 ( 3.03% ID/g, 4 h) at 0 and 5 µg spiking doses. In contrast, at 50 µg spiking dose the liver uptake (13.87 ( 1.43% ID/g, 4 h) was significantly reduced (P < 0.05). Tumor-to-liver ratio at 4 h thus increased from 0.62 ( 0.07 (0

Figure 4. Tumor-to-normal organ ratios of 64Cu-DOTA-ZEGFR:1907 in mice bearing the A431 tumor at 4 h p.i. (n ) 3).

µg spiking dose) and 0.56 ( 0.09 (5 µg spiking dose) to 1.32 ( 0.37 (50 µg spiking dose) (P < 0.05). Moreover, tumorto-blood ratios at 4 h also improved from 1.04 ( 0.01 (0 µg spiking dose) and 2.29 ( 0.43 (5 µg spiking dose) to 9.87 ( 2.99 (50 µg spiking dose) (P < 0.05) (Figure 4). The in vivo tumor targeting specificity was confirmed by coinjection of the probe with 500 µg of Ac-Cys-ZEGFR:1907. It was found that the tumor had a significantly reduced uptake of 6.15 ( 0.49% ID/g, compared with 17.35 ( 4.48% ID/g at 50 µg spiking dose (4 h p.i., P < 0.05). Liver uptake was also significantly decreased (6.47 vs 13.27% ID/g at 50 µg spiking dose, 4 h, P < 0.05). Small-Animal PET Imaging. Imaging studies were conducted at 1, 2, 4, and 24 h after tail vein injection of 64CuDOTA-ZEGFR:1907 with or without co-injection of 5, 50, and 500 µg (block) of cold Ac-Cys-ZEGFR:1907. Decay-corrected coronal microPET images are shown in Figure 5. At 5 µg spiking dose, the tumor was clearly visible up to 24 h p.i. with good tumorto-background contrast. Also, high activity accumulation in the kidneys and liver for 64Cu-DOTA-ZEGFR:1907 was observed (Figure 5E). At 0 and 50 µg spiking dose, tumors are both clearly visible at 4 h p.i., while much better tumor imaging contrast was obtained for 50 µg spiking dose at early time points (1 and 2 h p.i.) (Figure 5A and B). For the blocking group (500 µg), the tumor was barely visible on micro-PET images at all time points (Figure 5C). Quantification analysis of PET images


Miao et al.

Figure 5. Representative images of decay corrected coronal microPET images of nude mice bearing the A431 tumor on the right shoulder at 1, 2, and 4 h after tail vein injection of 64Cu-DOTA-ZEGFR:1907 (1.11-1.85 MBq, 30-50 µCi) spiked with 0 µg (A), 50 µg (B), and 500 µg (C) of Ac-Cys-ZEGFR:1907. Arrows indicate the location of tumors. Quantitative analysis of tumor uptakes at 1, 2, and 4 h p.i. (D) ROI was drawn on coronal images. Uptake was calculated with the mean uptake value (n ) 3). (E) Representative images of decay corrected coronal (top) and transaxial (bottom) microPET images of nude mice bearing the A431 tumor on the right shoulder at 2, 4, and 24 h after tail vein injection of 64 Cu-DOTA-ZEGFR:1907 (1.11-1.85 MBq, 30-50 µCi, spiked with 5 µg of Ac-Cys-ZEGFR:1907). Arrows indicate the location of tumors.

Figure 6. Representative western blot detection of EGFR expression in liver, tumor, and kidney tissue samples. Liver, A431 tumor, and kidney homogenates were prepared, and 50 µg of protein was detected with the rabbit antihuman EGFR monoclonal antibody.

showed much lower tumor uptake for mice injected with 500 µg spiking dose compared with 50 µg spiking dose (P < 0.05) at all time points (1-4 h p.i.) (Figure 5D). Western blot analysis is shown in Figure 6. High EGFR expression was detected for both liver and A431 tumor samples, which explained the blocking effects in the tumor and liver observed in microPET imaging studies.

DISCUSSION Molecular imaging techniques have been demonstrated to be powerful noninvasive tools to address many important clinical questions. PET imaging is particularly useful because of its high sensitivity, high spatial resolution, strong quantification ability, and its great potential for clinical translation. The EGFR targeted PET molecular probes are expected to provide a real-time assay of EGFR expression in all tumor sites (primary and metastatic lesions) in living subjects. These imaging probes could potentially be used for early detection of EGFR positive tumor recurrence, stratification of cancer patients and optimization of the dose for EGFR targeted therapy, and monitoring the efficacy of EGFR based tumor treatment.

Affibody scaffold proteins are chemically robust like small molecules. They have good in vivo stability and bioavailability (18-20). In this study, we site-specifically conjugated an Affibody analogue Ac-Cys-ZEGFR:1907 with a versatile chelator DOTA-monomaleimide and measured the binding specificity and affinity to EGFR. The conjugated DOTA-ZEGFR:1907 has good binding affinity with a KD of 20 nM. In vitro cell uptake experiments showed that 64Cu-DOTA-ZEGFR:1907 had rapid accumulation in the A431 cells. The uptake reached a plateau in 0.5 h. This accumulation is EGFR specific since blocking with cold DOTA-ZEGFR:1907 significantly reduced cell uptake from 26.77% ( 1.35% to 1.86% ( 0.67% of applied activity (1 h) (P < 0.05). It is reported that in EGFR over expression tumors the concentration of EGFR is about 100 nM (26, 27); thus, 20 nM affinity was considered efficient for the in vivo imaging application. Site-specifically labeled 64Cu-DOTA-ZEGFR:1907 showed good in vivo pharmacokinetics for EGFR targeted PET imaging. Good contrast imaging of the EGFR positive A431 tumor xenograft model were obtained with optimized spiking doses (5-50 µg) at early time (1 h p.i.) after the injection of 64Cu-DOTAZEGFR:1907. High tumor uptake (∼10% ID/g, 5 µg spiking dose) at 1 h p.i. confirmed the fast tumor targeting ability of the probe. The biodistribution study showed that optimal good tumor/blood ratio was obtained with partially blocking with 5-50 µg unlabeled Affibody (9.87 ( 2.99 at 4 h p.i., 50 µg spiking dose; 5.39 ( 0.69 at 24 h p.i., 5 µg spiking dose), which was consistent with reports that radioiodine labeled EGF with large (3 mg) spiking dose of cold EGF can give improved imaging quality in clinics (11).This observation may be attributed to the fact that the liver has high expression of EGFR (Figure 6) and could retain significant amount of the radiolabeled ligand (Figure 5). When spiked with an optimized amount of the cold ligand, the availability of the probe in the circulation would be increased. Because of the relatively small size of the scaffold protein, majority of the probe was quickly cleared through the kidney-urinary system. EGFR targeting specificity of 64CuDOTA-ZEGFR:1907 was also proven in vivo. When 500 µg of

64


Ac-Cys-ZEGFR:1907 was co-injected, uptakes in high EGFR expression organs/tissues, such as the tumor and liver, were both significantly reduced at 4 h p.i. (P < 0.05). High liver accumulation of radioactivity (>10% ID/g, 4 h p.i.) was also observed, which is mainly caused by the high EGFR expression in the liver, as well as nonspecific clearance of the probe through the hepatobiliary system and transchelation of 64 Cu in the liver. Recently, the Affibody ZEGFR:1907 has been modified with a chelator Bz-DTPA through lysine residues and then labeled with 111In (28). The resulting single photon emission computed tomography (SPECT) probe 111In-BzDTPA-ZEGFR:1907 showed much lower blood and liver uptakes (0.23 and 1.5% ID/g, repectively, at 4 h p.i., with 50 µg spiking dose). However, tumor uptake of 111In-Bz-DTPA-ZEGFR:1907 was also much lower (3-4% ID/g at 4 h p.i., with 50 µg spiking dose). This may be caused by the nonspecific labeling strategy used in the study, which could lead to reduced affinity of the probe and low tumor uptake. This is a disadvantage, especially because it may result in low sensitivity for the detection of the tumors with moderate or low EGFR expression, such as breast carcinoma (only about 3% of A431 EGFR expression level) (2). Further studies are required to compare these two EGFR imaging probes to conclude which one would be better for a specific application. 64Cu-DOTA-ZEGFR:1907 also showed extremely high kidney uptake (196 ( 4% ID/g vs 207.35 ( 101.16% ID/g at 4 h, with 50 µg spiking dose). Thus, radioactive coppers with higher fraction of positron emission and shorter half-life such as 60Cu (t1/2 ) 24.5 min, 93% β+) and 61Cu (t1/2 ) 3.333 h, 60% β+) will be more favorable for future clinical translation (29). 18F (t1/2 )109.8 min, 98% decay by positron emission) labeled Affibody could also have significantly reduced kidney radiation because of the nonresidualizing properties of radiohalogen (30). On the basis of the biodistribution and microPET imaging results, it has been found that Affibody molecule based probe 64 Cu-DOTA-ZEGFR:1907 shows much better properties for PET imaging than 64Cu labeled monoclonal antibody Cetuximab. Affibody protein ZEGFR:1907 is much smaller than Cetuximab (7 kDa vs 150 kDa). ZEGFR:1907 site specifically modified with DOTA still has low-nanomolar affinity to EGFR (IC50 ) 26.1 ( 8.8 nM). High tumor uptake (over 9% ID/g) could be obtained at as early as 1 h p.i. of the 64Cu-DOTA-ZEGFR:1907, while it takes ∼24 h for 64Cu labeled DOTA-Cetuximab to achieve over 10% ID/g tumor uptake (A431, U87MG, and PC-3 tumors) (31, 32). In addition, blood clearance was much faster for 64Cu-DOTA-ZEGFR:1907 (1.96 ( 0.30% ID/g at 50 µg spiking dose, 4 h p.i. vs 9.4 ( 1.2, 5.7 ( 1.9% ID/g for 64Cu-DOTAcetuximab at 16 and 48 h p.i., respectively); thus, much better contrast was obtained for the Affibody based PET probe at early time. Compared with Affibody protein based probes, antibody based PET probes have poorer tumor penetration and higher nonspecific uptake because of their bulky size. Recently, in a quantitative PET imaging study of EGFR using a 64Cu-labeled antibody, it was found that EGFR expression level measured in head and neck cancer cells was not correlated with the PET signal at all (33), suggesting that a bulky antibody based PET probe may not be suitable for quantitative imaging of EGFR. It is expected that an Affibody based PET probe such as 64CuDOTA-ZEGFR:1907 may overcome this problem. Further research is ongoing in our group to address this important question.

CONCLUSIONS In conclusion, a novel Affibody based PET probe, 64CuDOTA-ZEGFR:1907, has been successfully developed for molecular imaging of EGFR in vivo. This probe provides high specificity, sensitivity, and excellent tumor contrast as early as


1 h p.i.. It may find important applications in cancer PET molecular imaging.

ACKNOWLEDGMENT This work was supported, in part, by the California Breast Cancer Research Program 14IB-0091 and an SNM Pilot Research Grant.

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Small-Animal PET Imaging of Human Epidermal Growth Factor

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