All chemicals, unless otherwise noted, were acquired from Sigma-Aldrich (St. Louis, MO) and were used as received without further purification. All water employed was ultrapure (>18.2 MΩ cm–1
at 25 °C, Milli-Q, Millipore, Billerica, MA), and was passed through a 10 cm column of Chelex resin (Bio-Rad Laboratories, Hercules, CA) before use. DMSO was of molecular biology grade (>99.9%: Sigma, D8418), and all other solvents were of the highest grade commercially available. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid monoN-
hydroxysuccinimidylester (DOTA-NHS) was purchased from Macrocyclics Inc. (Dallas, TX). N
-Succinyldesferrioxamine B was prepared according to published procedures.(48
) All instruments were calibrated and maintained in accordance with standard quality-control procedures.(49
) UV–vis measurements were taken on a Cary 100 Bio UV–vis spectrophotometer. NMR spectroscopy was performed on a Bruker 500 MHz NMR with Topsin 2.1
software for spectrum analysis. HPLC was performed using a Shimadzu HPLC equipped with a C-18 reversed-phase column (Phenomenex Luna analytical 4.6 × 250 mm or SemiPrep 21.2 × 100 mm, 5 μm, 1.0 or 6.0 mL/min), 2 LC-10AT pumps, a SPD-M10AVP photodiode array detector, and a gradient of 0:100 MeCN/H2
O (both with 0.1% TFA) to 100:0 MeCN/H2
O over 15 min].
Cu was purchased from Washington University, St. Louis, where it was produced on the Washington University School of Medicine Cyclotron (model CS-15, Cyclotron Corp.) by the 64
Cu reaction and purified as previously described to yield [64
with an effective specific activity of 200–400 mCi/μg (7.4–14.8 GBq/μg).(50
Zr was produced at Memorial Sloan-Kettering Cancer Center on an EBCO TR19/9 variable-beam energy cyclotron (Ebco Industries Inc., British Columbia, Canada) via the 89
Zr reaction and purified in accordance with previously reported methods to yield 89
Zr with a specific activity of 5.28–13.43 mCi/μg (195–497 MBq/μg).(51
) All buffers used for 64
Cu and 89
Zr labeling were passed through Chelex resin before use. Activity measurements were made using a Capintec CRC-15R Dose Calibrator (Capintec, Ramsey, NJ). For accurate quantification of activities, experimental samples were counted for 1 min on a calibrated Perkin-Elmer (Waltham, MA) Automatic Wizard2
Gamma Counter. Both 64
Cu and 89
Zr labeling reactions were monitored using silica-gel impregnated glass-fiber instant thin layer chromatography paper (Pall Corp., East Hills, NY) and analyzed on a Bioscan AR-2000 radio-TLC plate reader using Winscan Radio-TLC software (Bioscan Inc., Washington, DC). Human breast cancer cell lines BT-474 and MDA-MB-468 were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were grown by serial passage.
Synthesis of 3-(4-Benzylamino)-1,2,4,5-tetrazine (Tz)
The protocol from Deveraj et al. was employed for the synthesis with slight modifications.(24
) 4-(Aminomethyl)-benzonitrile hydrochloride (0.84 g, 0.005 mol) formamidine acetate (2.08 g, 0.02 mol), and elemental sulfur (0.16 g, 0.005 mol) were added to a dry, 50 mL round-bottom flask. Anhydrous hydrazine (2 mL) was then added to the flask, and the resultant orange reaction mixture was stirred for 20 h. After the allotted time, 1% HCl(aq)
(50 mL) was slowly added to the reaction mixture, and the resultant solution was stirred for 10 min and subsequently filtered through a medium glass frit. The remaining orange solution was cooled in an ice bath to 0 °C, and a solution of 1.7 g of NaNO2
in 15 mL of water was then added dropwise to the reaction mixture. While still cooled in an ice bath, acetic acid (50 mL) was added slowly, and the reaction mixture immediately turned bright pink. After allowing this solution to warm to room temperature over the course of 3 h, the solvent was evaporated at 50 °C and 20 Torr on a rotary evaporator. The resultant red crude solids were dissolved in 250 mL of water with 0.1% TFA. The aqueous solution was adsorbed onto a C18
column (Waters C18
Sep-Pak, Waters Corp., Milford, MA), washed with copious amounts of water, and eluted with acetonitrile. This bright pink, organic solution was evaporated to dryness, and the red crude was purified by flash chromatography (CombiFlash automated chromatography system, Teledyne Isco Inc., Lincoln, NE) using a gradient of 100% CHCl3
(0.01% TFA) from 0 to 4 min followed by 0:100 MeOH (0.01%TFA)/CHCl3
(0.01% TFA) to 30:70 (0.01%TFA)/HCl3
(0.01% TFA) over 16 min. After the removal of solvent, the pure product was obtained in 35% yield (0.33 g, 0.0018 mol). 1
H NMR (500 MHz, D2
O), δ, ppm: 10.46 (s, 1H), 8.54 (d, 2H), 7.77 (d, 1H), 4.41 (s, 2H). ESI-MS: 188.1 [M+H]+
. HPLC tR
= 7.1 min.
Synthesis of N1-(5-(4-((4-(1,2,4,5-Tetrazin-3-yl)benzyl)amino)-4-oxobutanamido)pentyl)-N1-hydroxy-N4-(5-(N-hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4-oxobutanamido)pentyl)succinamide (Tz-DFO)
3-(4-Benzylamino)-1,2,4,5-tetrazine (8 mg, 0.045 mmol) was dissolved in DMSO (3 mL), and diisopropylethylamine (16 μL, 0.09 mmol) was added to this solution. After 15 min of stirring at RT, the pink DMSO solution was added to a second, premixed solution of N-succinyldesferrioxamine B (60 mg, 0.09 mmol) and benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP, 53 mg, 0.12 mmol) in DMSO (3 mL). The combined reaction was stirred overnight and subsequently purified via C18 cartridge (Waters C18 Sep-Pak, Waters Corp., Milford, MA) and semipreparative reverse-phase HPLC. The purified product was obtained in 50% yield (molecular weight = 852.9, 19 mg, 0.023 mmol). 1H NMR (500 MHz, DMSO-d6), δ, ppm: 10.59 (s, 1H), 9.64 (s, 1H), 9.59 (s, 1H), 8.49 (m, 1H), 8.44 (d, 2H), 7.9–7.7 (m, 3H), 7.51 (d, 2H), 4.44 (d, 2H), 3.5–3.5 (m, 6H), 3.05–2.95 (m, 6H), 2.55 (t, 4H), 2.45–2.35 (m, 4H), 2.25 (t, 4H), 1.97 (s, 3H), 1.52–1.48 (m, 6H), 1.40–1.36 (m, 6H), 1.23–1.20 (m, 6H). ESI-MS: 831.5 [M+H]+, 853.6 [M+Na]+. HPLC tR = 10.2 min.
Synthesis of 2,2′,2″-(10-(2-((4-(1,2,4,5-Tetrazin-3-yl)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Tz-DOTA)
3-(4-Benzylamino)-1,2,4,5-tetrazine (20 mg, 0.12 mmol) was dissolved in PBS (5 mL, pH 8.5), and diisopropylethylamine (40 μL, 0.24 mmol) was added to this solution. This solution was then added to solid DOTA-NHS (50 mg, 0.065 mmol), and the resultant solution was stirred overnight at room temperature. The reaction was subsequently purified via C18 cartridge (Waters C18 Sep-Pak, Waters Corp., Milford, MA) and semipreparative reverse-phase HPLC. The purified product was obtained in 62% yield (molecular weight = 573.6, 23 mg, 0.04 mmol). 1H NMR (500 MHz, DMSO-d6), δ, ppm: 10.61 (s, 1H), 9.11 (br s, 1H), 8.50 (d, 2H), 7.62 (d, 2H), 4.50 (s, 2H), 4.42–4.38 (m, 4H), 3.65 (br s, 4H), 10.61 (s, 1H), 3.65–3.55 (m, 8H), 3.18–3.14 (m, 8H). ESI-MS: 574.5 [M+H]+, 596.1 [M+Na]+, 612.2 [M+K]+. HPLC tR = 8.1 min.
A protocol similar to that published by Devaraj et al. was employed for antibody modification.(24
) 5-Norbornene-2-carboxylic acid (40 mg, 0.29 mmol) was incubated with 1.3 equiv of disuccinimidyl carbonate (100 mg, 0.39 mmol) and 1 equiv of pyridine (23 mg, 0.29 mmol) in dry acetonitrile (3 mL) for 2 h at room temperature. After 2 h, the solvent was removed via rotary evaporation, and the crude norbornene-succinimidyl ester product was recovered. Trastuzumab (purchased commercially as Herceptin, Genentech, San Francisco, CA) was purified using centrifugal filter units with a 30
000 molecular weight cutoff (Amicon Ultra 4 Centrifugal Filtration Units, Millipore Corp., Billerica, MA) and phosphate buffered saline (PBS, pH 7.4) to remove α–α-trehalose dihydrate, l
-histidine, and polysorbate 20 additives. After purification, the antibody was taken up in PBS pH 8.0. Subsequently, 300 μL of antibody solution (150–250 μM) were combined with 100 μL PBS pH 8.0 and 1.5, 3, or 5 equiv of the crude norbornene-NHS ester in 10 μL of either DMF or DMSO. The reaction was incubated at room temperature for 2 h, followed by centrifugal filtration to purify the resultant antibody conjugate.
To perform the chelator ligation, 100 μL antibody solution (75–150 μM, PBS pH 7.4) was combined with 200 μL buffer (PBS pH 7.4) and a 10-fold molar excess of either Tz-DOTA or Tz-DFO in 10 μL DMSO (molar excess calculated based on initial norbornene reaction stoichiometry). The reaction was incubated at RT for 5 h and subsequently purified using centrifugal filtration to yield the completed DOTA- and DFO-modified antibodies. The final bioconjugates were stored in PBS pH 7.4 at 4 °C.
Labeling of DOTA-T/N-trastuzumab with 64Cu
DOTA-T/N-trastuzumab (0.2–0.3 mg) was added to 200 μL labeling buffer (50 mM NH4OAc, pH 5.5, though 50 mM NaOAc, pH 5.5 also is sufficient). [64Cu]CuCl2 (29.6–37 MBq, 800–1000 μCi) in approximately 1–3 μL 0.1 M HCl were then added to the antibody solution, and the resultant solution was incubated at room temperature for 1 h. After 1 h, the reaction progress was assayed using ITLC with an eluent of 50 mM EDTA, pH 5. The resultant 64Cu-DOTA-T/N-trastuzumab was purified using either size-exclusion chromatography (Sephadex G-25 M, PD-10 column, 30 kDa, GE Healthcare; dead volume = 2.5 mL, eluted with 200 mL fractions of PBS, pH 7.4) or centrifugal column filtration. The radiochemical purity of the final radiolabeled bioconjugate was assayed by radio-TLC and was found to be >99% in all preparations. In the ITLC experiments, 64Cu-DOTA-T/N-trastuzumab remains at the baseline, while 64Cu2+ ions and [64Cu]Cu-EDTA elute with the solvent front.
Labeling of DFO-T/N-trastuzumab with 89Zr
DFO-T/N-trastuzumab (0.2–0.3 mg) was added to 200 μL buffer (PBS, pH 7.5). [89Zr]Zr-oxalate (29.6–37 MBq, 800–1000 μCi) in 1.0 M oxalic acid was adjusted to pH 7.2–8.5 with 1.0 M Na2CO3. After evolution of CO2(g) stops, the 89Zr solution was added to the antibody solution, and the resultant mixture was incubated at room temperature for 1 h. After 1 h, the reaction progress was assayed using ITLC with an eluent of 50 mM EDTA, pH 5. The resultant 89Zr-DFO-T/N-trastuzumab was purified using either size-exclusion chromatography (Sephadex G-25 M, PD-10 column, 30 kDa, GE Healthcare; dead volume = 2.5 mL, eluted with 200 mL fractions of PBS, pH 7.4) or centrifugal column filtration. The radiochemical purity of the final radiolabeled bioconjugate was assayed by radio-TLC and was found to be >99% in all preparations. In the ITLC experiments, 89Zr-DFO-T/N-trastuzumab remains at the baseline, while 89Zr4+ ions and [89Zr]-EDTA elute with the solvent front.
The number of accessible DFO and DOTA chelates conjugated to the antibodies was measured by radiometric isotopic dilution assays following methods similar to those described by Anderson et al. and Holland et al.34,42,52,53
All experiments were performed in triplicate.
The immunoreactivity of the 64
Cu-DOTA- and 89
Zr-DFO-T/N-trastuzumab bioconjugates was determined using specific radioactive cellular-binding assays following procedures derived from Lindmo et al.54,55
To this end, BT-474 cells were suspended in microcentrifuge tubes at concentrations of 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, and 1.0 × 10 6
cells/mL in 500 μL PBS (pH 7.4). Aliquots of either 64
Cu-DOTA- or 89
Zr-DFO-T/N-trastuzumab (50 μL of a stock solution of 10 μCi in 10 mL of 1% bovine serum albumin in PBS pH 7.4) were added to each tube (n
= 4; final volume: 550 μL), and the samples were incubated on a mixer for 60 min at room temperature. The treated cells were then pelleted via centrifugation (3000 rpm for 5 min), resuspended, and washed twice with cold PBS before removing the supernatant and counting the activity associated with the cell pellet. The activity data were background-corrected and compared with the total number of counts in appropriate control samples. Immunoreactive fractions were determined by linear regression analysis of a plot of (total/bound) activity against (1/[normalized cell concentration]). No weighting was applied to the data, and data were obtained in triplicate.
The stability of the 64Cu-DOTA- and 89Zr-DFO-T/N-trastuzumab bioconjugates with respect to radiochemical purity and loss of radioactivity from the antibody was investigated in vitro by incubation of the antibodies in human serum for 48 h (64Cu) or 7 d (89Zr) at room temperature and 37 °C. The radiochemical purity of the antibodies was determined via radio-TLC with an eluent of 50 mM EDTA pH 5.0 (vide supra).
Human breast cancer cell lines BT474 and MDA-MB-468 were obtained from the American Tissue Culture Collection (HTB-20 and HTB-132, respectively, ATCC, Bethesda, MD) and maintained in a 1:1 mixture of Dulbecco’s Modified Eagle medium: F-12 medium, supplemented with 10% heat-inactivated fetal calf serum (Omega Scientific, Tarzana, Ca), 2.0 mM glutamine, nonessential amino acids, and 100 units/mL penicillin, and 100 units/mL streptomycin in a 37 °C environment containing 5% CO2. Cell lines were harvested and passaged weekly using a formulation of 0.25% trypsin/0.53 mM EDTA in Hank’s Buffered Salt Solution without calcium and magnesium.
All experiments were performed under an Institutional Animal Care and Use Committee-approved protocol, and the experiments followed institutional guidelines for the proper and humane use of animals in research. Six- to eight-week-old Athymic nu/nu female mice (NCRNU-M) were obtained from Taconic Farms Incorporated (Hudson, NY). Animals were housed in ventilated cages, were given food and water ad libitum, and were allowed to acclimatize for approximately 1 week prior to treatment. Prior to tumor inoculation, mice were subcutaneously implanted with 0.72 mg 60 day release 17β-estradiol pellets (SE-121, Innovative Research of America, Sarasota, Florida) using a 10 gauge trocar. After several days, BT474 tumors were induced on the right shoulder by a subcutaneous injection of 3.0 × 106 cells in a 100 μL cell suspension of a 1:1 mixture of fresh media/BD Matrigel (BD Biosciences, Bedford, Ma). MDA-MB-468 tumors were induced on the left shoulder by a subcutaneous injection of 2.0 × 106 cells in the same manner (the number of cells injected was varied as described to compensate for cell growth rates and thus provide approximately the same tumor size at the time of radiopharmaceutical injection).
Acute in vivo biodistribution studies were performed in order to evaluate the uptake of the 64Cu-DOTA- and 89Zr-DFO-conjugated antibodies in mice bearing bilateral, subcutaneous BT-474 and MDA-MB-468 tumors (100–150 mm3, 4 weeks postinoculation). Mice were randomized before the study and were warmed gently with a heat lamp for 5 min before administration of 64Cu-DOTA-T/N-trastuzumab (0.74–1.11 MBq [20–30 μCi] in 200 μL 0.9% sterile saline) or 89Zr-DFO-T/N-trastuzumab (0.56–0.74 MBq [15–20 μCi] in 200 μL 0.9% sterile saline) via intravenous tail vein injection (t = 0). Animals (n = 4 per group) were euthanized by CO2(g) asphyxiation at 6, 12, 24, 36, 48, and 72 h (64Cu) or 6, 24, 48, 72, 96, and 120 h (89Zr). After asphyxiation, 13 organs (including both tumors) were removed, rinsed in water, dried in air for 5 min, weighed, and counted in a gamma counter calibrated for either 64Cu or 89Zr. Counts were converted into activity using a calibration curve generated from known standards. Count data were background- and decay-corrected to the time of injection, and the percent injected dose per gram (%ID/g) for each tissue sample was calculated by normalization to the total activity injected.
Small-Animal PET Imaging
PET imaging experiments were conducted on either a microPET Focus 120 (89
Zr) or a microPET R4 (64
Cu) rodent scanner (Concorde Microsystems).(56
) Mice bearing bilateral, subcutaneous BT-474 (right shoulder) and MDA-MB-468 (left shoulder) tumors (100–150 mm3
, 4 weeks postinoculation) were administered 64
Cu-DOTA-T/N-trastuzumab (11.1–12.9 MBq [300–345 μCi] in 200 μL 0.9% sterile saline) or 89
Zr-DFO-T/N-trastuzumab (10.7–11.8 MBq [290–320 μCi] in 200 μL 0.9% sterile saline) via intravenous tail vein injection (t
= 0). Approximately 5 min prior to the acquisition of PET images, mice were anesthetized by inhalation of 2% isoflurane (Baxter Healthcare, Deerfield, IL)/oxygen gas mixture and placed on the scanner bed; anesthesia was maintained using 1% isoflurane/gas mixture. PET data for each mouse were recorded via static scans at various time points between 6 and 120 h. A minimum of 20 million coincident events were recorded for each scan, which lasted between 10 and 45 min. An energy window of 350–700 keV and a coincidence timing window of 6 ns were used. Data were sorted into 2D histograms by Fourier rebinning, and transverse images were reconstructed by filtered back-projection (FBP) into a 128 × 128 × 63 (0.72 × 0.72 × 1.3 mm3
) matrix. The image data were normalized to correct for nonuniformity of response of the PET, dead-time count losses, positron branching ratio, and physical decay to the time of injection, but no attenuation, scatter, or partial-volume averaging correction was applied. The counting rates in the reconstructed images were converted to activity concentrations (percentage injected dose [%ID] per gram of tissue) by use of a system calibration factor derived from the imaging of a mouse-sized water-equivalent phantom containing 64
Cu or 89
Zr. Images were analyzed using ASIPro VM
software (Concorde Microsystems).
Labeling Norbornene-Trastuzumab with [64Cu]-Tz-DOTA
Tz-DOTA (5 μL of 1 mM solution in DMSO) was added to labeling buffer (50 mM NH4
OAc, pH 5.5), and [64
(40.7–55.5 MBq [1100–1500 μCi]) in 0.1 M HCl were added to the reaction mixture. The resultant solution was incubated for 1 h at 85 °C, followed by purification via C18
cartridge (Waters C18
Sep-Pak, Waters Corp., Milford, MA) and radiochemical purity analysis via analytical HPLC (tR
= 10 min). The purified, radiolabeled [64
Cu]-Tz-DOTA was then added to a solution of norbornene-modified trastuzumab (0.4 mg, initial reaction stoichiometry of 5:1 norbornene/mAb) in PBS pH 7.4. The reaction mixture was allowed to incubate at 37 °C for 3 h. After 3 h, the progress of the reaction was assayed with radio-TLC using an eluent of 50 mm EDTA pH 5.0, and the radiolabeled antibody was purified with centrifugal filtration using centrifugal filter units with a 30
000 molecular weight cutoff (Amicon Ultra 4 Centrifugal Filtration Units, Millipore Corp., Billerica, MA) and phosphate buffered saline (PBS, pH 7.4). The radiochemical purity of the final radiolabeled bioconjugate was assayed again by radio-TLC and was found to be >99% in all preparations. In the radio-TLC experiments, 64
Cu-DOTA-T/N-trastuzumab remains at the baseline, while 64
Cu]Cu-Tz-DOTA, and [64
Cu]Cu-EDTA elute with the solvent front.
Data were analyzed by the unpaired, two-tailed Student’s t test. Differences at the 95% confidence level (P < 0.05) were considered to be statistically significant.