The Effect of Bi-Terminal PEGylation of an Integrin α_vβ₆–Targeted¹⁸F 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 4-18F-fluorobenzoic acid (18F-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 18F-FBAA20FMDV2-PEG28 (4) and 18F-FBA-PEG28-A20FMDV2-PEG28 (5), on αvβ6-targeted tumor uptake and pharmacokinetics. The results are compared with 18F-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 18F-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.

Solid-phase synthesis and fluorine-18 radiolabeling of cycloRGDyK

“…[25–27] Additionally, SPPS provides a pseudodilution effect which can aid with cyclization, and permit radiolabeling of small amounts of peptide, a current challenge of fluorine-18 radiochemistry. In addition, the solid phase synthetic strategy is amenable to automation.…”

“…The generated [ 18 F]FBA was quenched with hydrochloric acid, cartridge purified (C18-SepPak), concentrated, and added to the 5 mg of pre-swelled peptidyl resin ( 8 ) (0.49 mg, 0.79 μmol of peptide ( 8 )). [27] Following our well established solid-phase radiolabeling procedure for linear peptides, [27] a brief 30 minute coupling of [ 18 F]FBA (96–150 mCi) to 8 via activation of [ 18 F]FBA in situ by HATU (5 mg) and DIPEA (10 μL) and a short 30 minute cleavage/deprotection with TFA-TIPS mixture, generated cRGDyK([ 18 F]FBA) ( 10 ) in 14 ± 2% (n = 4) decay corrected yield. Compound 10 was obtained in >99% radiochemical purity after purification by reverse-phase HPLC in a synthesis time of 90 minutes from [ 18 F]FBA.…”

Recent advances in the bioanalytical methods of polyethylene glycols and PEGylated pharmaceuticals

“…The shielding effect of PEG was later extended to protect PEG‐peptide conjugates and low MW PEG was also used. In the work of Hausner et al., the 20‐residue linear peptide A20FMDV2, which exhibits high selectivity and affinity to the tumor‐related αvβ6 integrin, was evaluated with the help of the 4‐ 18 F‐fluorobenzoic acid ( 18 F‐FBA) radiotracers [96]. After intravenous injection administration to tumor‐bearing mice, the biodistribution was analyzed by PET/CT imaging and γ‐counter.…”

Fully automated peptide radiolabeling from [18F]fluoride

“…[13][14][15][16][17][18][19] Another member of the integrin family of receptors that is currently being investigated is the integrin-a v b 6 , an epithelial-specic cell surface receptor that is undetectable in healthy adults, but is signicantly up-regulated in a wide range of epithelial derived cancers including, pancreas, colon, non-small cell lung cancer, ovarian, breast, prostate, and oral squamous cell carcinoma. [20][21][22][23][24][25][26] Sutcliffe et al, have focused signicant effort on the development of a v b 6 -targeting peptides based on the 20 amino acid sequence derived from an envelope protein of the foot-and-mouth disease virus. [23][24][25][26] Another example are the gastrin releasing peptide receptors (GRPRs) that are overexpressed in many cancers, including breast, pancreatic, and prostate, with GRPR-mediated signalling being linked to several oncogenic processes such as invasiveness and proliferation.…”

“…[20][21][22][23][24][25][26] Sutcliffe et al, have focused signicant effort on the development of a v b 6 -targeting peptides based on the 20 amino acid sequence derived from an envelope protein of the foot-and-mouth disease virus. [23][24][25][26] Another example are the gastrin releasing peptide receptors (GRPRs) that are overexpressed in many cancers, including breast, pancreatic, and prostate, with GRPR-mediated signalling being linked to several oncogenic processes such as invasiveness and proliferation. 27,28 These receptors have been targeted by a series of bombesin peptides for both imaging and therapeutic purposes with their efficacy being examined in several clinical studies.…”