title: An engineered knottin peptide labeled with 18F for PET imaging of integrin expression.
abstract: Knottins are small constrained polypeptides that share a common disulfide-bonded framework and a triple-stranded beta-sheet fold. Previously, directed evolution of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin led to the identification of a mutant that bound to tumor-specific alpha(v)beta(3) and alpha(v)beta(5) integrin receptors with low nanomolar affinity. The objective of this study was to prepare and evaluate a radiofluorinated version of this knottin (termed 2.5D) for microPET imaging of integrin positive tumors in living subjects. Knottin peptide 2.5D was prepared by solid-phase synthesis and folded in vitro, and its free N-terminal amine was reacted with N-succinimidyl-4-18/19F-fluorobenzoate (18/19F-SFB) to produce the fluorinated peptide 18/19F-FB-2.5D. The binding affinities of unlabeled knottin peptide 2.5D and 19F-FB-2.5D to U87MG glioblastoma cells were measured by competition binding assay using 125I-labeled echistatin. It was found that unlabeled 2.5D and 19F-FB-2.5D competed with 125I-echistatin for binding to cell surface integrins with IC(50) values of 20.3 +/- 7.3 and 13.2 +/- 5.4 nM, respectively. Radiosynthesis of 18F-FB-2.5D resulted in a product with high specific activity (ca. 100 GBq/micromol). Next, biodistribution and positron emission tomography (PET) imaging studies were performed to evaluate the in vivo behavior of 18F-FB-2.5D. Approximately 3.7 MBq 18F-FB-2.5D was injected into U87MG tumor-bearing mice via the tail vein. Biodistribution studies demonstrated that 18F-FB-2.5D had moderate tumor uptake at 0.5 h post injection, and coinjection of a large excess of the unlabeled peptidomimetic c(RGDyK) as a blocking agent significantly reduced tumor uptake (1.90 +/- 1.15 vs 0.57 +/- 0.14%ID/g, 70% inhibition, P < 0.05). In vivo microPET imaging showed that 18F-FB-2.5D rapidly accumulated in the tumor and quickly cleared from the blood through the kidneys, allowing excellent tumor-to-normal tissue contrast to be obtained. Collectively, 18F-FB-2.5D allows integrin-specific PET imaging of U87MG tumors with good contrast and further demonstrates that knottins are excellent peptide scaffolds for development of PET probes with potential for clinical translation.
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Bio-X Program, Stanford University, Stanford, California 94305, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Miao
authors.forename: Gang
authors.initials: G
authors.lastname: Ren
authors.forename: Hongguang
authors.initials: H
authors.lastname: Liu
authors.forename: Richard H
authors.initials: RH
authors.lastname: Kimura
authors.forename: Lei
authors.initials: L
authors.lastname: Jiang
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
authors.forename: Sanjiv Sam
authors.initials: SS
authors.lastname: Gambhir
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cheng
mesh_terms: D000818:Animals; D005462:Fluorine Radioisotopes / Q000737:chemistry; D006801:Humans; D016023:Integrins / Q000032:analysis* / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D009374:Neoplasms, Experimental; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D049268:Positron-Emission Tomography; D015202:Protein Engineering; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
chemical_list: D005462:Fluorine Radioisotopes; D016023:Integrins; D010455:Peptides