Unless otherwise specified, all reagents were analytical grade and were obtained from commercial sources. The ZHER2:342-Cys Affibody molecules were kindly provided by our Cooperative Research and Development Agreement (CRADA) partner in Sweden (Affibody AB; http://www.affibody.com). 18F radionuclide was obtained from the NIH/CC cyclotron facility from proton irradiation of O-18-enriched water. Trastuzumab (Herceptin) was purchased from Genentech, San Francisco, CA, USA, as a lyophilized white powder and reconstituted with the supplied solvent. Pentamethylbenzyl (4-trimethylammonium) benzoate trifluoromethanesulfonate was prepared as described previously .
Synthesis of N-(2-(4-[18F]fluorobenzamido)ethyl)maleimide ([18F]FBEM)
[18F]Fluorobenzoic acid was prepared as previously described using a manual synthesis method . Briefly, pentamethylbenzyl (4-trimethylammonium) benzoate trifluoromethanesulfonate (3 mg, 6 µmol) was heated for 10 min in a sealed V-vial with [18F]fluoride (550 to 2,220 MBq) in the presence of Kryptofix (2.2.2; 6 µmol) and K2CO3 (3 µmol) in CH3CN (0.1 ml). The intermediate was treated with trifluoroacetic acid (TFA) to provide 4-[18F]fluorobenzoic acid. The 4-[18F]fluorobenzoic acid was treated with N-(2-aminoethyl)maleimide (1.52 mg, 6 µmol), diethyl cyanophosphonate (1.3 mg, 8 µmol), and N,N-diisopropylethyl amine (10 ml). The resulting solution was heated at 70°C for 5 min. This solution was diluted with 150 µl water and injected onto an high-performance liquid chromatography (HPLC) column [Luna C-18 (Phenomenex), 10 × 250 mm, 20% CH3CN, 80% water, 5 ml/min]. The product eluting at about 17 min was collected, diluted to about 20 ml with water and trapped on a BondElut C-18 (500 mg). The product was eluted from the column with CH2Cl2; the organic layer was carefully separated from the small amount of water and carefully evaporated into the bottom of a 1.5-ml Eppendorf tube.
Conjugation of N-2-(4-[18F]fluorobenzamido)ethyl]maleimide with ZHER2:342−Cys ([18F]FBEM-ZHER2:342-Affibody)
The isolated [18F]FBEM (111-370 MBq) was dissolved in 10 µl of CH3CN. In the meantime, stock Affibody molecule ZHER2:342-Cys (100 µg in 100 µl phosphate-buffered saline, PBS; 12 nmol) was treated with 10 µl of 1 M dithiothreitol (DTT) for 40 min at 37°C. The solution was then eluted in 0.25 ml fractions of 0.1 M NaOAc, 0.1% ascorbic acid, pH 7, from a NAP-5 column (GE Healthcare Bio-Science AB, Uppsala, Sweden; cut-off, 5 kDa). Fraction 4 (0.25 ml) from this column was added to the solution of [18F]FBEM. This solution was incubated for 15-30 min at room temperature and loaded onto a NAP-5 column. The NAP-5 column was eluted with 250 µl portions of PBS. The most concentrated fraction containing the radiolabeled protein (fraction 4) was collected and used for the biological experiments. The radiochemical yield of 18F-labeled Affibody conjugate using this un-optimized procedure was approximately 5% based on starting 18F-fluoride and uncorrected for decay. The total procedure requires about 2 h.
Protein concentration of the labeled preparation was determined by measuring UV absorbance at 280 nm (NanoDrop Technologies, Wilmington, DE, USA) using the conversion 1 AU = 0.99 mg/ml.
HPLC analysis of [18F]FBEM-ZHER2:342-Affibody
Analysis of the protein product was conducted using a Vydac protein C-4 column (4.5 × 150 mm). Gradient elution was employed (5-75% of 0.1% TFA in acetonitrile versus 0.1% TFA in water over 15 min). [18F]FBEM-ZHER2:342-Affibody molecules were eluted at about 8 min.
Human parental breast (SKBR-3, MCF7) and ovarian (SKOV-3) cancer cell lines that express different levels of HER2, as well as a HER2-negative human glioblastoma cell line (U251), were purchased from the American Type Culture Collection (Rockville, MD, USA). The breast cancer cell line, stably transfected with HER2 (MCF7/HER2-18), was kindly provided by Dr. John W. Park, University of California, San Francisco, CA, USA. The cells were cultured in DMEM/F12 medium (SKBR-3, SKOV-3, U251) or in DMEM (MCF7, MCF7/HER2-18) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (GIBCO, Grand Island, NY, USA) and penicillin/streptomycin (100 U/ml of each). Cells were grown as a monolayer at 37°C in a humidified atmosphere containing 5% CO2. In the case of MCF7/HER2 transfectants, the culture medium also contained 400 µg/ml Geneticin, G418 (GIBCO, Grand Island, NY, USA).
In vitro cell-binding assays
The in vitro binding characteristics of the [18F]FBEM-ZHER2:342-Affibody molecules were assessed using saturation and displacement cell-binding assays. The day before the experiments, cells were seeded in six-well plates at concentration of 2.5 × 105 cells per ml. For receptor saturation analysis, the cells were incubated (4°C, 2 h) with increasing concentrations (3.3-420 nM) of [18F]FBEM-ZHER2:342-Affibody alone or with additional 100-fold excess of non-labeled Affibody molecules. Then, the incubation medium was collected, and the cells were washed with cold PBS and trypsinized, and the cell-associated radioactivity was measured using a γ-counter (1480 Wizard 3, Automatic Gamma Counter, Perkin-Elmer, Waltham, MA, USA). The total non-specific and specific binding were plotted against the total molar concentration of added [18F]FBEM-ZHER2:342-Affibody, and the data were analyzed by non-linear regression using GraphPad Prism (GraphPad Software, San Diego, CA, USA)
For the displacement assay, the cells were incubated with 216 nM of [18F]FBEM-ZHER2:342-Affibody and increasing concentrations (1.2 nM-120 µM) of non-labeled Affibody molecules. The rest of the experimental procedures were the same as those used for the saturation studies.
The specificity of binding was also checked for cell lines that express different numbers of HER2 receptors. The experiments were carried out at the same way as for saturation analysis; however, in this case, only one concentration of [18F]FBEM-ZHER2-Affibody was chosen. In addition, some wells were preincubated (30 min) with 100-fold excess amount of unlabeled Affibody or trastuzumab (1 µg/ml).
Five- to 7-week-old female athymic nude mice were injected subcutaneously with 1 × 107 of SKOV-3 or with 106 of U251 cells suspended in Matrigel (BD Bioscience, San Jose, CA, USA). Tumors were placed on the hind leg or on the back. These locations were chosen to optimize the PET images by placing the tumor well away from the radioactivity in major body structures and reduce background radioactivity. U251 and SKOV-3 tumors (50-250 mg) developed after 2 and 4-5 weeks, respectively. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institute of Health Guide for the Care and Use of Animals on approved studies from the National Institutes of Health Institutional Animal Care and Use Committee.
Athymic nude mice bearing SKOV-3 tumors were injected with 1.8-2.2 MBq (1.6-2 µg, 100 µl) of [18F]FBEM-ZHER2-Affibody into the tail vein. Groups of three to six mice were sacrificed, and their organs dissected 1, 2, 3, 4, and 6 h post-injection. Then, blood, tumor, and major organs were collected and weighed. The radioactivity in the tissues was measured along with a standard of the injected dose using γ-counter. The results were calculated as percentage injected dose per gram of tissue (% ID/g).
To study the blood kinetics and the stability of [18F]FBEM-ZHER2:342-Affibody in vivo, the blood was collected at 1-6 h post-injection in vials containing 30 µl heparin. The samples were weighed, and the radioactivity was measured by γ-counter. Then, the blood was centrifuged (10 min at 10,000×g) and fractions of plasma (200-300 µl) were separated on NAP-5 columns. The mean concentrations of radioactivity in the blood (% ID/g) were plotted against time after injection, and the curve was fitted by single exponential decay. Similar procedure was used to analyze high molecular weight (HMW)/low molecular weight (LMW) ratio of radioactivity in the urine collected 60 min post-injection.
To confirm the specificity of binding in vivo, one group of animals bearing HER2-negative U251 tumors was injected with the [18F]FBEM-ZHER2:342-Affibody and another group bearing HER2-positive SKOV-3 tumors was pre-injected with 400 µg of non-labeled Affibody 45 min before the [18F]FBEM-ZHER2:342-Affibody was injected. Two hours later, all animals were sacrificed, and their organs analyzed as described above.
Mice were anesthetized using isoflurane/O2 (1.5-5% v/v) and injected with approximately 3.7-4.4 MBq (3.4-4 µg, 100 µl) of the [18F]FBEM-ZHER2:342-Affibody via the tail vein. PET scans were performed using the Advanced Technology Laboratory Animal Scanner (ATLAS) PET scanner . Whole-body (1, 2, 3, 4, and 6 h; four bed positions, each 15 min) and dynamic data acquisition (1 frame, 10 min, up to 12 frames) were started about 2 min after radiotracer injection and recorded with a 100-700 keV energy window. The images were reconstructed by a two-dimensional ordered-subsets expectation maximum (2D-OSEM) algorithm, and no correction was applied for attenuation or scatter. For each scan, regions of interest were drawn over the tumor, normal tissue, and major organs. The maximum radioactivity accumulation within the tumor or organs was obtained from mean pixel values within the multiple region of interest (ROI). The results were calculated as a percentage injected dose per gram (% ID/g) and standard uptake value (SUV). At the end of the study, an 18F source of known activity was imaged to obtain MBq of 18F per counts per second for the imaging system (calibration factor). Then, every ROI (counts per second per cubic centimeter) was multiplied by this factor and divided by injected activity.