Characterization and evaluation of ⁶⁴Cu-labeled A20FMDV2 conjugates for imaging the integrin α_vβ₆

Purpose: The integrin α_vβ₆ 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 α_vβ ₆ 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 α_vβ ₆-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers ⁶⁴Cu-CB-TE1A1P-1 and ⁶⁴Cu-BaBaSar-1 showed improved specificity for the α_vβ₆ 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.