Characterization and Evaluation of 64Cu-Labeled A20FMDV2 Conjugates for Imaging the Integrin αvβ6

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 64Cu 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 64Cu 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 64Cu-CB-TE1A1P-1 and 64Cu-BaBaSar-1 showed improved specificity for the αvβ6 positive tumor in vivo over 64Cu-DOTA-1 and 64Cu-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, 64Cu-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 64Cu-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.

Comparative Evaluation of Anti-HER2 Affibody Molecules Labeled with 64Cu Using NOTA and NODAGA

“…Still, comparison of 64 Cu-labeled NOTA- and DOTA-conjugated agonistic bombesin analogs reveals lower tumor and renal radioactivity retention when using NOTA [ 24 ], which is in agreement with our present data. Very interesting is an evaluation of 64 Cu-labeled A20FMDV2 peptide conjugates for imaging the integrin α v β 6 , since radiometal-labeled A20FMDV2 has high renal uptake [ 42 ]. In that study, the distribution of radioactivity after injection of 64 Cu-NOTA-A20FMDV2 had the same features as distribution after injection of 64 Cu-NOTA-ZHER2:S1, that is, rapid release of radioactivity from kidneys, poor retention of radioactivity in tumors, and an increase in hepatic uptake between 1 and 4 hours after injection, with a subsequent decrease at 24 h. Overall, increasing the time between 64 Cu-NOTA-ZHER2:S1 or 64 Cu-NODAGA-ZHER2:S1 injection and imaging did not improve imaging contrast and did not show any advantage over the use of 68 Ga-NODAGA-ZHER2:S1.…”

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display

“…68 Ga-DOTA-SFITGv6 accumulates in the tumor but not in inflammatory lesions of patients PET/CT scans in a compassionate use setting were performed in two tumor patients after application of 68 Ga-DOTA-SFITGv6 and 18 F-FDG, respectively (Fig. 6).…”

“…The RGD motif occurs in many extracellular matrix ligands of integrins; however, the motif DLXXL was identified by phage display within 7-and 12-residue peptides as a ITGa v b 6 -specific binding motif (14,15). Because ITGa v b 6 is overexpressed in many carcinomas associated with poor prognosis including lung, pancreatic, ovarian, colorectal and cervical cancers, but only at low or undetectable levels in normal tissues (16) it might represent an important target for imaging and anticancer therapies (17)(18)(19)(20)(21)(22)(23).…”

PET Radiopharmaceuticals for Imaging Integrin Expression: Tracers in Clinical Studies and Recent Developments

“…A study by Hu et al [43] was designed to determine the best candidate out of four chelating systems to label PEG 28 -A20FMDV2 with 64 Cu. This include a triazacyclononane derivative (NOTA), a tetraazacyclododecane derivative (DOTA), a tetraazabicyclo[6.6.2] hexadecane derivative (CB-TE1A1P), and a hexaazabicyclo[6.6.6]icosane derivative (BaBaSar).…”