title: Pharmacokinetically stabilized cystine knot peptides that bind alpha-v-beta-6 integrin with single-digit nanomolar affinities for detection of pancreatic cancer.
abstract: OBJECTIVE
  Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin α(v)β(6), a cell surface receptor being evaluated as a novel clinical biomarker.
  
  
  METHODS
  To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3-6 nmol/L) to integrin α(v)β(6). The binders do not cross-react with related integrin α(v)β(5), integrin α(5)β(1), or tumor-angiogenesis-associated integrin, α(v)β(3).
  
  
  RESULTS
  Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin α(v)β(6). Clinically relevant tumor-to-muscle ratios of 7.7 ± 2.4 to 11.3 ± 3.0 were achieved within 1 hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting α(v)β(6)-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3).
  
  
  CONCLUSIONS
  We have engineered highly stable cystine knot peptides with potent and specific integrin α(v)β(6)-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging.
authors.affiliation: Canary Center for Cancer, Early Detection, Molecular Imaging Program, Department of Radiology, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Kimura
authors.forename: Robert
authors.initials: R
authors.lastname: Teed
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hackel
authors.forename: Marybeth A
authors.initials: MA
authors.lastname: Pysz
authors.forename: Courtney Z
authors.initials: CZ
authors.lastname: Chuang
authors.forename: Ataya
authors.initials: A
authors.lastname: Sathirachinda
authors.forename: Jürgen K
authors.initials: JK
authors.lastname: Willmann
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D057005:Bioengineering; D014408:Biomarkers, Tumor / Q000032:analysis; D057771:Cystine-Knot Miniproteins / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics*; D005260:Female; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D010190:Pancreatic Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D049268:Positron-Emission Tomography / Q000379:methods; D011485:Protein Binding; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D014018:Tissue Distribution
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D057771:Cystine-Knot Miniproteins; D016023:Integrins; D019275:Radiopharmaceuticals; C073878:integrin alphavbeta6