title: 18F-fluorobenzoate-labeled cystine knot peptides for PET imaging of integrin αvβ6.
abstract: Integrin αvβ6 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 αvβ6 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 αvβ6, for (18)F radiolabeling and PET imaging of BxPC3 pancreatic adenocarcinoma xenografts in mice. Cystine knot peptides were labeled with N-succinimidyl-4-(18)F-fluorobenzoate and evaluated for binding affinity and serum stability. Peptides conjugated with (18)F-fluorobenzoate (2-3 MBq) were injected via the tail vein into nude mice xenografted with BxPC3 (integrin αvβ6-positive) or 293 (integrin αvβ6-negative) tumors. Small-animal PET scans were acquired at 0.5, 1, and 2 h after injection. Ex vivo γ-counting of dissected tissues was performed at 0.5 and 2 h.
  
  
  RESULTS
  (18)F-fluorobenzoate peptides were produced in 93% ((18)F-fluorobenzoate-R01) and 99% ((18)F-fluorobenzoate-S02) purity. (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-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. (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-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 = 4-5). Target specificity was confirmed by low tumor uptake in integrin αvβ6-negative 293 tumors (1.4 ± 0.6 and 0.5 ± 0.2 %ID/g, respectively, for (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02; both P < 0.05; n = 3-4) and low muscle uptake (3.1 ± 1.0 and 2.7 ± 0.4 tumor to muscle for (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02, respectively). Small-animal PET data were corroborated by ex vivo γ-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 (18)F-fluorobenzoate-R01 and (18)F-fluorobenzoate-S02; n = 8). Uptake in healthy pancreas was low (0.3% ± 0.1% for (18)F-fluorobenzoate-R01 and 0.03% ± 0.01% for (18)F-fluorobenzoate-S02; n = 8).
  
  
  CONCLUSIONS
  These cystine knot peptide tracers, in particular (18)F-fluorobenzoate-R01, show translational promise for molecular imaging of integrin αvβ6 overexpression in pancreatic and other cancers.
authors.affiliation: Department of Radiology, Molecular Imaging Program at Stanford, Canary Center for Cancer Early Detection, Stanford University, Stanford, California, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hackel
authors.forename: Richard H
authors.initials: RH
authors.lastname: Kimura
authors.forename: Zheng
authors.initials: Z
authors.lastname: Miao
authors.forename: Hongguang
authors.initials: H
authors.lastname: Liu
authors.forename: Ataya
authors.initials: A
authors.lastname: Sathirachinda
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cheng
authors.forename: Frederick T
authors.initials: FT
authors.lastname: Chin
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D001565:Benzoates / Q000493:pharmacokinetics*; D045744:Cell Line, Tumor; D005260:Female; D016023:Integrins / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D008657:Metabolic Clearance Rate; D051379:Mice; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods*; D009928:Organ Specificity; D010190:Pancreatic Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism*; D049268:Positron-Emission Tomography / Q000379:methods*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014018:Tissue Distribution
chemical_list: D000951:Antigens, Neoplasm; D001565:Benzoates; D016023:Integrins; D019275:Radiopharmaceuticals; C073878:integrin alphavbeta6; C022893:4-fluorobenzoic acid
keywords: cystine knot; integrin αvβ6; positron emission tomography