Characterization and evaluation of (64)Cu-labeled A20FMDV2 conjugates for imaging the integrin αvβ 6

Abstract

Purpose: The integrin αvβ6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for (64)Cu radiolabeling of A20FMDV2, an αvβ6 targeting peptide.

Procedures: Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4'-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with (64)Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted.

Results: All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37-50 °C for 15 min) with high specific activity (0.58-0.60 Ci/μmol). All radiotracers demonstrated αvβ6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers (64)Cu-CB-TE1A1P-1 and (64)Cu-BaBaSar-1 showed improved specificity for the αvβ6 positive tumor in vivo over (64)Cu-DOTA-1 and (64)Cu-NOTA-1 (+/- tumor uptake ratios-3.82 +/- 0.44, 3.82 ± 0.41, 2.58 ± 0.58, and 1.29 ± 0.14, respectively). Of the four radiotracers, (64)Cu-NOTA-1 exhibited the highest liver uptake (10.83 ± 0.1 % ID/g at 4 h).

Conclusions: We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear "best candidate" for the (64)Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.

Fig. 1

The four chelators conjugated to PEG₂₈-A20FMDV2.

Fig. 2

In vitro binding (black) and internalization (gray) results after 60 min using α_vβ₆-positive and α_vβ₆-negative cells. Internalization levels shown as percent relative to total activity.

Fig. 3

a Serum stability HPLC traces for each radiotracer after incubating in mouse serum for 1, 4, and 24 h (top to bottom for each radiotracer); b urine metabolite HPLC traces after 1 and 4 h p.i. for each radiotracer (unable to collect urine sample for ⁶⁴Cu-NOTA-1 at 4 h p.i.).

Fig. 4

Uptake in the (+) and (−) tumors as determined by biodistribution studies at 1, 4, and 24 h p.i. for a ⁶⁴Cu-CB-TE1A1P-1, b ⁶⁴Cu-DOTA-1, c ⁶⁴Cu-NOTA-1, and d ⁶⁴Cu-BaBaSar-1 (individually scaled). These values were used to calculated e positive to negative tumor uptake ratios. Uptake values are represented as % ID/g. P values were determined using paired, two-tailed Student t tests.

Fig. 5

a Blocking study results with an injection of NH₂-PEG₂₈-A20FMDV2 10 min prior to injecting the formulated radiotracers. Uptake in the key organs b kidney and c liver at 1, 4, and 24 h p.i.; d tumor to blood and e tumor to muscle ratios at 1, 4, and 24 h p.i. Uptake values are represented as % ID/g with n=3/radiotracer per experiment per time point.

Fig. 6

a Reconstructed 3D PET/CT images showing (+) (green arrow) and (−) (red arrow) tumors. Mice were anesthetized using 2–3 % isoflurane and received 150–250 µCi of formulated radiotracer via tail vein. All images were acquired 4 h p.i. using 20 min static scans. b Autoradiography slices (20 µm) of (+) and (−) tumors, sectioned at 4 h p.i. and exposed overnight. Each slice read at a 50-µm resolution. c Histology slices (5 µm) from (+) and (−) tumors after immunohistochemistry staining for α_vβ₆ viewed at ×4 magnification. Scale bar = 400 µm.