¹⁸F-fluorobenzoate-labeled cystine knot peptides for PET imaging of integrin α_vβ₆

Integrin α_vβ₆ is a cell surface receptor minimally expressed by healthy tissue but elevated in lung, colon, skin, ovarian, cervical, and pancreatic cancers. A molecular PET agent for integrin α_vβ₆ could provide significant clinical utility by facilitating both cancer staging and treatment monitoring to more rapidly identify an effective therapeutic approach. Methods: Here, we evaluated 2 cystine knot peptides, R01 and S02, previously engineered with a 3-6 nM affinity for integrin α_vβ₆, for ¹⁸F radiolabeling and PET imaging of BxPC3 pancreatic adenocarcinoma xenografts in mice. Cystine knot peptides were labeled with N-succinimidyl-4- ¹⁸Ffluorobenzoate and evaluated for binding affinity and serum stability. Peptides conjugated with ¹⁸F-fluorobenzoate (2-3 MBq) were injected via the tail vein into nude mice xenografted with BxPC3 (integrin α_vβ₆-positive) or 293 (integrin α_vβ₆-negative) tumors. Small-animal PET scans were acquired at 0.5, 1, and 2 h after injection. Ex vivo g-counting of dissected tissues was performed at 0.5 and 2 h. Results: ¹⁸F- fluorobenzoate peptides were produced in 93% (¹⁸F-fluorobenzoate-R01) and 99% (¹⁸F-fluorobenzoate-S02) purity. ¹⁸F- fluorobenzoate-R01 and ¹⁸Ffluorobenzoate- S02 had affinities of 1.1 ± 0.2 and 0.7 ± 0.4 nM, respectively, and were 87% and 94%, respectively, stable in human serum at 37°C for 2 h. ¹⁸F- fluorobenzoate-R01 and ¹⁸Ffluorobenzoate- S02 exhibited 2.3 ± 0.6 and 1.3 ± 0.4 percentage injected dose per gram (%ID/g), respectively, in BxPC3 xenografted tumors at 0.5 h (n 5 4-5). Target specificity was confirmed by low tumor uptake in integrin α_vβ ₆-negative 293 tumors (1.4 ± 0.6 and 0.5 ± 0.2 %ID/g, respectively, for ¹⁸F-fluorobenzoate-R01 and ¹⁸F- fluorobenzoate-S02; both P<0.05; n 5 3-4) and low muscle uptake (3.1 ± 1.0 and 2.7 ± 0.4 tumor to muscle for ¹⁸F-fluorobenzoate-R01 and ¹⁸F-fluorobenzoate-S02, respectively). Small-animal PET data were corroborated by ex vivo g-counting of dissected tissues, which demonstrated low uptake in nontarget tissues with only modest kidney uptake (9.2 ± 3.3 and 1.9 ± 1.2 %ID/g, respectively, at 2 h for ¹⁸F- fluorobenzoate-R01 and ¹⁸Ffluorobenzoate- S02; n 5 8). Uptake in healthy pancreas was low (0.3% ± 0.1% for ¹⁸F-fluorobenzoate- R01 and 0.03% ± 0.01% for ¹⁸F-fluorobenzoate-S02; n 5 8). Conclusion: These cystine knot peptide tracers, in particular ¹⁸F-fluorobenzoate-R01, show translational promise for molecular imaging of integrin α_vβ₆ overexpression in pancreatic and other cancers.