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

Abstract

Radiotracers based on the peptide A20FMDV2 selectively target the cell surface receptor integrin αvβ6. 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-(18)F-fluorobenzoic acid ((18)F-FBA), we have shown that the introduction of poly(ethylene glycol) (PEG) improves pharmacokinetics, including increased uptake in αvβ6-expressing tumors. The present study evaluated the effect of site-specific C-terminal or dual (N- and C-terminal) PEGylation, yielding (18)F-FBA-A20FMDV2-PEG28 (4) and (18)F-FBA-PEG28-A20FMDV2-PEG28 (5), on αvβ6-targeted tumor uptake and pharmacokinetics. The results are compared with (18)F-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, DX3puroβ6 (αvβ6+), DX3puro (αvβ6-), and BxPC-3 (αvβ6+), we evaluated the radiotracers in vitro (serum stability; cell binding and internalization) and in vivo in mouse models bearing paired DX3puroβ6-DX3puro and, for 5, BxPC-3 xenografts.

Results: The size and location of the PEG units significantly affected αvβ6 targeting and pharmacokinetics. Although the C-terminally PEGylated 4 showed some improvements over the un-PEGylated (18)F-FBA-A20FMDV2 (1), it was the bi-terminally PEGylated 5 that displayed the more favorable combination of high αvβ6 affinity, selectivity, and pharmacokinetic profile. In vitro, 5 bound to αvβ6-expressing DX3puroβ6 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, αvβ6-directed binding in the paired DX3puroβ6-DX3puro model (1 h: DX3puroβ6, 2.3 ± 0.2 percentage injected dose per gram [%ID/g]; DX3puroβ6/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 αvβ6+ tumor uptake and retention.

Keywords: PEGylation; integrin αvβ6; metabolism; peptide; positron emission tomography.

Figure 1

Structures of radiotracers evaluated.

Figure 2

Binding and internalization of radiotracers in vitro. (A) Paired, integrin α_vβ₆–expressing DX3puroβ6 cell line and non–α_vβ₆-expressing DX3puro control (P ≤ 0.0001 for corresponding data sets). (B) Integrin α_vβ₆–expressing BxPC-3 cell line. Filled columns = fraction of total radioactivity (n = 4/radiotracer/cell line/condition; 60 min); bars = SD. Data for 1–3 are from Hausner et al. (28).

Figure 3

Biodistribution data of ¹⁸F-FBA-A20FMDV2-PEG₂₈ (4) in mice bearing paired α_vβ₆-expressing DX3puroβ6 and non–α_vβ₆-expressing DX3puro xenograft tumors. (A) Organ uptake (%ID/g; bars = SD; n = 3/time point). *P ≤ 0.02 for corresponding time points. (B) Uptake ratios of 4 for tumors and selected organs (bars = SD). Bl = bladder.

Figure 4

Biodistribution data of ¹⁸F-FBA-PEG₂₈-A20FMDV2-PEG₂₈ (5) in mice bearing either paired α_vβ₆-expressing DX3puroβ6 and non–α_vβ₆-expressing DX3puro xenograft tumors or α_vβ₆-expressing BxPC-3 xenograft tumors. (A) Organ uptake (%ID/g; bars = SD; tumors: n = 3/time point, nontumor tissues: n = 6/time point). **P ≤ 0.001 for corresponding time points. ***P ≤ 0.014 for corresponding time points. Uptake ratios of 5 for tumors and selected organs in paired DX3 tumor model (B) and BxPC-3 tumor model (C) (bars = SD). Bl = bladder.

Figure 5

(A) Representative transaxial and coronal cross-sections of PET/CT images obtained after injection of ¹⁸F-FBA-PEG₂₈-A20FMDV2-PEG₂₈ (5; 8.9 MBq) in mouse bearing BxPC-3 xenograft (66 mg; arrow). CT is gray and PET red. B = Bladder; K = Kidneys. (B) Autoradiography image of BxPC-3 tumor harvested 1 h after injection of 5 (39 MBq; left) and matched adjacent immunohistochemistry slice stained for integrin α_vβ₆ expression (middle, right: magnified section).