Evaluation of [64Cu]Cu-DOTA and [64Cu]Cu-CB-TE2A Chelates for Targeted Positron Emission Tomography with an αvβ6-Specific Peptide

Significant upregulation of the integrin αvβ6 has been described as a prognostic indicator in several cancers, making it an attractive target for tumor imaging. This study compares variants of a PEGylated αvβ6-targeting peptide, bearing either an [>18F]fluorobenzoyl prosthetic group ([18F]FBA-PEG-A20FMDV2) or different [64Cu]copper chelators (DOTA-PEG-A20FMDV2, CB-TE2A-PEG-A20FMDV2). The compounds were evaluated in vitro by enzyme-linked immunosorbent assay (against the integrin αvβ6 and the closely related integrin αvβ6) and by cell labeling (αvβ6-positive DX3puroβ6/αvβ6-negative DX3puro) and in vivo using micro-positron emission tomography in a mouse model bearing paired DX3puroβ6/Dx3puro xenografts. In vitro, all three compounds showed excellent αvβ6-specific binding (50% inhibitory concentration [IC50](αvβ6) = 3 to g nmol/L; IC50(αvβ3) > 10 (μmol/L). In vivo, they displayed comparable, preferential uptake for the αvβ6-expressmg xenograft over the αvβ6-negative control (> 4:1 ratio at 4 hours postinjection). Whereas [64Cu]Cu-DOTA-PEG-A20FMDV2 resulted in increased levels of radioactivity in the liver, [64Cu]Cu-CB-TE2A-PEG-A20FMDV2 did not. Significantly, both 64Cu-labeled tracers showed unexpectedly high and persistent levels of radioactivity in the kidneys (> 40% injected dose/g at 4 and 12 hours postinjection). The findings underscore the potential influence of the prosthetic group on targeted in vivo imaging of clinically relevant markers such as αvβ6. Despite identical targeting peptide moiety and largely equal in vitro behavior, both 64Cu-labeled tracers displayed inferior pharmacokinetics, making them in their present form less suitable candidates than the 18F-labeled tracer for in vivo imaging of αvβ6

Reverse-phase high-pressure liquid chromatography (HPLC) was used to purify and analyze the compounds: solvent A, 0.05% trifluoroacetic acid (TFA) in water (v/v); solvent B, acetonitrile. HPLC systems were equipped with both an ultraviolet (UV) absorbance detector (UV 220 nm) and a radioactivity detector (photomultiplier tube [PMT]). The detectors were connected in series, resulting in a slight difference in retention times observed for radioactive compounds and their corresponding cold standards: microPET scanner (Siemens Medical Solutions USA, Malvern, PA), and data were processed with the accompanying ASIpro software.
Nonradioactive FBA or DOTA tris(t-Bu ester) was coupled to the N-terminus of the peptide on solid phase using a threefold excess of the acid and HATU/DIEA activation for 1 hour. CB-TE2A was preactivated 18 with N,N'-diisopropylcarbodiimide (DIC) in the presence of DIEA for 30 minutes at room temperature before being coupled to the peptide on solid phase.
The progress of the coupling reactions was monitored by the ninhydrin-based Kaiser test 23 or the picrylsulfonic acid test. 24 Side-chain protecting groups were removed and the peptides were cleaved off the solid support using TFA/1,2-ethanedithiol/triisopropyl-silane/ water 94:2.5:1: The crude peptides, dissolved in water, were washed three times with diethyl ether before purification on semipreparative HPLC (system B) and characterization using mass spectrometry.

Radiochemical Synthesis of [ 18 F]FBA-1
[ 18 F]FBA was prepared according to a published procedure and coupled to the N-terminally deprotected peptide on solid phase as previously described. 12,21,25 Briefly, the [ 18 F]FBA in DMF (50 µL) was withdrawn into a 1 mL fritted syringe containing the peptide resin (5 mg) swollen in DMF, followed by HATU (5 mg) in DMF (30 µL) and DIEA (10 µL) in DMF (30 µL). The reaction mixture was shaken at room temperature for 30 minutes. The solvent was removed, and the resin was washed with DMF (3 × 0.5 mL) and methanol (3 × 0.5 mL). The TFA cleavage mixture (0.7 mL; TFA/triisopropylsilane/water 95:2.5:2.5 v/v/v) was drawn into the syringe, and the reaction mixture was incubated at 30°C for 20 minutes. The solution was collected. Following evaporation of the cleavage solvents, the product was purified by semipreparative HPLC (system C). The fractions containing the product were collected, diluted with water to a total volume of 20 mL, and passed through a C18 Sep-Pak cartridge. The product, trapped on the cartridge, was washed with water (5 mL) and eluted with 1% acetic acid in ethanol (v/v). The solvent was removed by a stream of nitrogen and the product was reconstituted in phosphate-buffered saline (PBS), followed by pH adjustment. The specific activity was determined by comparison of the radiotracer with the nonradioactive standard (HPLC, system A). [ 64 Cu]Cu-DOTA-1 was purified by C18 solid-phase extraction (ORTG Inc.) and eluted in ethanol (400 µL). The solution was concentrated to a small volume (approximately 50 µL) using a vacuum-centrifuge and then formulated in PBS. The radiotracer was evaluated for radiochemical purity (TLC, HPLC) and specific activity (HPLC).

Radiochemical Synthesis of [ 64 Cu]Cu-CB-TE2A-1
The synthesis was similar to that of [ 64 Cu]Cu-DOTA-1 except the reaction solution was buffered to pH 7.5 to 8.5 and incubated at 90 to 95°C. Formulation and evaluation (TLC, HPLC) were performed as with [ 64 Cu]Cu-DOTA-1.

In Vitro Experiments
IC 50 values were determined as previously described 12 using competitive ELISA of the nonradioactive compounds FBA-1, DOTA-1, CB-TE2A-1, FBA-2, and the parent peptide A20FMDV2 (2) against biotinylated natural ligands, either biotinylated fibronectin (α v β 6 assays) or vitronectin (α v β 3 assays), in triplicate on microtiter plates. For cell binding experiments, 0.2 µCi aliquots of the radiotracer in 50 µL serum-free DMEM (pH 7.2) was added to the cell suspension (3.75 × 10 6 cells in 50 µL serum-free DMEM) and incubated at room temperature for 1 hour. The assay tubes, pretreated with bovine serum albumin (5% wt/v in PBS) to block nonspecific binding, were regularly agitated to prevent settling of the cells. Following gentle centrifugation, the supernatant was removed and the cells were washed with 0.5 mL serum-free DMEM. The supernatants were combined, and the cells were resuspended in 0.6 mL serum-free DMEM. To determine the fraction of radioactivity bound to cells, levels of radioactivity in the cell suspension and in the supernatants were measured in a gamma counter. For each radiotracer, experiments with DX3puroβ6 and DX3puro cell lines were carried out simultaneously (n ≥ 4/cell line).

In Vivo Experiments
All animal experiments were conducted under a protocol approved by the University of California, Davis, Animal Use and Care Committee. Male athymic mice (nu/nu; Charles River Laboratories, Wilmington, MA) were inoculated subcutaneously on opposite flanks in the shoulder region with 3 × 10 6 DX3puro or DX3puroβ6 cells in 100 µL serum-free DMEM. Cell lines were analyzed by flow cytometry before injection to confirm levels of integrin expression. Food and water were available ad libitum. Imaging was conducted 2 to 4 weeks after injection once tumors had reached a diameter of approximately 0.5 cm.

MicroPET Imaging
The radiotracer (approximately 100-250 µCi) in isotonic solution (150-200 µL) was injected intravenously into the tail vein via a catheter in mice (n = 3/tracer) anesthetized with 3% isoflurane. The animals were placed in a feet-first, prone position on the scanner bed and maintained under 1.5 to 2.0% isoflurane. Full body image reconstructions were obtained using a maximum a posteriori algorithm (ASIpro software).

Biodistribution
The radiotracer (approximately 15-30 µCi for the 1-, 2-, and 4-hour time points; 100 µCi for the 12-hour time point) in isotonic solution (150-200 µL) was injected intravenously into the tail vein via a catheter in mice anesthetized with 3% isoflurane. Following a conscious uptake period, the mice were anesthetized (4% isoflurane), sacrificed, and dissected (n = 3/ tracer). Organs were rapidly collected, and radioactivity was measured in a gamma counter. Calibrated, decay-corrected radioactivity concentrations are expressed as the percentage of injected dose per gram of tissue (% ID/g).

Statistical Analysis
Data are reported as mean ± SD. Two-tailed Student t-tests were performed to evaluate statistical significance, where p < .05 was considered statistically significant.

Nonradioactive Peptides
The compounds were prepared as described and obtained in high purity. The analytic data for the peptides are as follows: FBA-1,

In Vitro Evaluation: ELISA
Assays were performed to evaluate the ability of non-radioactive FBA-1, DOTA-1, and CB-TE2A-1 to inhibit binding of biotinylated natural ligands (fibronectin or vitronectin) to the immobilized integrin α v β 6 or the closely related α v β 3 . For comparison, the unPEGylated FBA-2 and the parent peptide A20FMDV2 (2) were included as well. Binding values are listed in Table 1. For all compounds, the data show IC 50 values in the low nanomolar range for integrin α v β 6 , together with no significant binding (IC 50 > 10 µmol/L) for integrin α v β 3 .

Discussion
The peptide A20FMDV2 has been recognized as a high-affinity, high-specificity ligand for the integrin α v β 6 12,26 and provides an attractive platform for the development of targeted in vivo imaging tracers. For the present study, solid-phase peptide synthesis and radiolabeling of the 18 F and 64 Cu tracers followed robust protocols, 15,21,25 yielding the radiotracers in high radiochemical purity (≥ 99%).
Given that modifications of a peptide such as the N-terminal addition of the prosthetic group and/or PEGylation can potentially influence the targeted binding behavior, the affinities and selectivities for α v β 6 of FBA-1, DOTA-1, and CB-TE2A-1 were first compared with A20FMDV2 (2) 12 and unPEGylated FBA-2 (see Figure 1). The results obtained by competitive ELISA (see Table 1) demonstrated that neither the introduction of the PEG moiety nor the choice of prosthetic group had any significant deleterious effect on binding to purified α v β 6 . All compounds selectively bound to integrin α v β 6 with the same high affinity as the parent A20FMDV2 and exhibited > 1,600-fold selectivity over the closely related integrin α v β 3 . These findings were corroborated by subsequent cell binding studies with the radiotracers using the melanoma cell line DX3puroβ6, stably transfected to express α v β 6 , and its paired control line DX3puro (see Figure 2): 44 to 54% of [ 18 F]FBA-1, [ 64 Cu]Cu-DOTA-1, and [ 64 Cu]Cu-CB-TE2A-1 bound to DX3puroβ6 within 1 hour, whereas less than 5% bound to DX3puro under identical conditions. Notably, both cell lines expressed comparable levels of integrins α v β 3 and other integrins, 12 underscoring the high selectivity of the A20FMDV2 peptide sequence for α v β 6 . Preliminary studies with the 18 F tracers indicated some internalization into cells (not shown). PEGylation is known to positively affect the characteristics of biologically important drug or diagnostic molecules. 27,28 In this cell binding assay, the presence of a small PEG spacer between the prosthetic group and the targeting peptide was also found to have a significant, positive effect on binding to α v β 6 , whereas an additional change of the prosthetic group at the end of the PEG spacer had only a slight effect: only 14% of the unPEGylated [ 18 F]FBA-2 bound to DX3puroβ6, compared with the 54% for its PEGylated derivative, [ 18 F]FBA-1, and 53% and 44% for [ 64 Cu]Cu-DOTA-1 and [ 64 Cu]Cu-CB-TE2A-1, respectively. These results highlight the significant differences between α v β 6 captured on microtiter plates and α v β 6 expressed on a cell surface. They underscore the importance of considering the biologic environment early in the development of targeted imaging tracers.
In preliminary studies, [ 18 F]FBA-1 had shown the greatest promise in vivo, combining generally good clearing from nontarget tissues with excellent retention in α v β 6 -expressing xenografts. Besides improved tumor retention, introduction of the PEG moiety also resulted in elevated levels of radioactivity in the kidneys, especially at the early time points, but renal clearing remained efficient (see Figure 4D): values dropped from 24.7 ± 4.8% ID/g at 1 hour post injection to 2.7 ± 0.8% ID/g at 4 hours postinjection (compared with 3.6 ± 1.4% ID/g and 0.15 ± 0.09% ID/g for [ 18 F]FBA-2 at the same time points). 12 13,29 It is generally understood that glomerular filtration and reabsorption in the proximal tubules (endocytosis and lysosomal metabolism) play key roles in the retention and that different mechanisms may be involved to various degrees for different radiotracers. To reduce trapping of the radiotracer or its metabolites in the tubular cells and thus reduce the radiation dose to the kidney, either the rate of endocytosis needs to be low or exocytosis and subsequent excretion in the urine need to be efficient. 29,30 In some instances, reducing the positive charge of metal-chelating radiotracers resulted in significantly improved clearance behavior, whereas the correlation was less clear in other studies. 29,31,32 When charge effects were evaluated specifically for 64 Cu-labeled macrocyclic complexes, increased positive formal charges were also found to result in increased kidney accumulation, whereas the related negatively charged or neutral complexes showed better renal clearance. 33  Clearly, this assumption in not supported by our observations. Similar, seemingly contradictory results have been reported for charge modifications of some octreotide radiotracers. 13,29 Anderson and colleagues showed that radiometal chelate-conjugated peptides that bind to cell surface receptors such as somatostatin are taken up intracellularly into lysosomes, and then the compounds are metabolized to radiometal chelate-N-terminal amino acids. 34,35 These metal chelate-based metabolites are retained in the kidneys, whereas halogenated metabolites are likely effluxed from the kidney cells. Interestingly, PEGylation has been suggested as a general strategy to improve the pharmacokinetics of radiopharmaceuticals and thereby reduce renal retention. 14,27,29 Despite the unfavorable renal pharmacokinetics encountered for [ 64 Cu]Cu-DOTA-1 and [ 64 Cu]Cu-CB-TE2A-1, some noteworthy differences were observed for other organs. Most notably, the CB-TE2A tracer was superior to the DOTA tracer with respect to liver uptake (see Figure 4).
Experiments were performed in triplicate, and values are expressed as mean ± SD (where appropriate).
* Data from Hausner SH et al. 12 Mol Imaging. Author manuscript; available in PMC 2015 January 08.