The Effect of Bi-Terminal PEGylation of an Integrin αvβ6-Targeted 18F Peptide on Pharmacokinetics and Tumor Uptake

Radiotracers based on the peptide A20FMDV2 selectively target the cell surface receptor integrin alpha(v)beta(6). This integrin has been identified as a prognostic indicator correlating with the severity of disease for several challenging malignancies. In previous studies of A20FMDV2 peptides labeled with 4-F-18-fluorobenzoic acid (F-18-FBA), we have shown that the introduction of poly(ethylene glycol) (PEG) improves pharmacokinetics, including increased uptake in alpha(v)beta(6)-expressing tumors. The present study evaluated the effect of site-specific C-terminal or dual (N-and C-terminal) PEGylation, yielding F-18-FBA-A20FMDV2-PEG(28) (4) and F-18-FBA-PEG(28)-A20FMDV2-PEG(28) (5), on alpha(v)beta(6)-targeted tumor uptake and pharmacokinetics. The results are compared with F-18-FBA-labeled A20FMDV2 radiotracers (1-3) bearing either no PEG or different PEG units at the N terminus. Methods: The radiotracers were prepared and radiolabeled on solid phase. Using 3 cell lines, DX3puro beta 6 (alpha v beta 6+), DX3puro (alpha(v)beta(6)-),and BxPC-3 (alpha v beta 6+), we evaluated the radiotracers in vitro (serum stability; cell binding and internalization) and in vivo in mouse models bearing paired DX3puro beta 6-DX3puro and, for 5, BxPC-3 xenografts. Results: The size and location of the PEG units significantly affected alpha(v)beta(6) targeting and pharmacokinetics. Although the C-terminally PEGylated 4 showed some improvements over the un-PEGylated F-18-FBA-A20FMDV2 (1), it was the bi-terminally PEGylated 5 that displayed the more favorable combination of high alpha(v)beta(6) affinity, selectivity, and pharmacokinetic profile. In vitro, 5 bound to alpha(v)beta(6)-expressing DX3puro beta 6 and BxPC-3 cells with 60.5% +/- 3.3% and 48.8% +/- 8.3%, respectively, with a significant fraction of internalization (37.2% +/- 4.0% and 37.6% +/- 4.1% of total radioactivity, respectively). By comparison, in the DX3puro control 5 showed only 3.0% +/- 0.5% binding and 0.9% +/- 0.2% internalization. In vivo, 5 maintained high, alpha(v)beta(6)-directed binding in the paired DX3puro beta 6-DX3puro model (1 h: DX3puro beta 6, 2.3 +/- 0.2 percentage injected dose per gram [%ID/g]; DX3puro beta 6/DX3puro ratio, 6.5: 1; 4 h: 10.7:1). In the pancreatic BxPC-3 model, uptake was 4.7 +/- 0.9 % ID/g (1 h) despite small tumor sizes (20-80 mg). Conclusion: The bi-PEGylated radiotracer 5 showed a greatly improved pharmacokinetic profile, beyond what was predicted from individual N-or C-terminal PEGylation. It appears that the 2 PEG units acted synergistically to result in an improved metabolic profile including high alpha(v)beta(6)+ tumor uptake and retention.