title: Selective Imaging of VEGFR-1 and VEGFR-2 Using 89Zr-Labeled Single-Chain VEGF Mutants.
abstract: Vascular endothelial growth factor-A (VEGF-A) acts via 2 vascular endothelial growth factor receptors, VEGFR-1 and VEGFR-2, that play important and distinct roles in tumor biology. We reasoned that selective imaging of these receptors could provide unique information for diagnostics and for monitoring and optimizing responses to anticancer therapy, including antiangiogenic therapy. Herein, we report the development of 2 first-in-class89Zr-labeled PET tracers that enable the selective imaging of VEGFR-1 and VEGFR-2.
  
  
  METHODS
  Functionally active mutants of scVEGF (an engineered single-chain version of pan-receptor VEGF-A with an N-terminal cysteine-containing tag for site-specific conjugation), named scVR1 and scVR2 with enhanced affinity to, respectively, VEGFR-1 and VEGFR-2, were constructed. Parental scVEGF and its receptor-specific mutants were site-specifically derivatized with the89Zr chelator desferroxamine B via a 3.4-kDa PEG linker.89Zr labeling of the desferroxamine B conjugates furnished scV/Zr, scVR1/Zr, and scVR2/Zr tracers with high radiochemical yield (>87%), high specific activity (≥9.8 MBq/nmol), and purity (>99%). Tracers were tested in an orthotopic breast cancer model using 4T1luc-bearing syngeneic BALB/c mice. For testing tracer specificity, tracers were coinjected with an excess of cold proteins of the same or opposite receptor specificity or pan-receptor scVEGF. PET imaging, biodistribution, and dosimetry studies in mice, as well as immunohistochemical analysis of harvested tumors, were performed.
  
  
  RESULTS
  All tracers rapidly accumulated in orthotopic 4T1luc tumors, allowing for the successful PET imaging of the tumors as early as 2 h after injection. Blocking experiments with an excess of pan-receptor or receptor-specific cold proteins indicated that more than 80% of tracer tumor uptake is VEGFR-mediated, whereas uptake in all major organs is not affected by blocking within the margin of error. Critically, blocking experiments indicated that VEGFR-mediated tumor uptake of scVR1/Zr and scVR2/Zr was mediated exclusively by the corresponding receptor, VEGFR-1 or VEGFR-2, respectively. In contrast, uptake of pan-receptor scV/Zr was mediated by both VEGFR-1 and VEGFR-2 at an approximately 2:1 ratio.
  
  
  CONCLUSIONS
  First-in-class selective PET tracers for imaging VEGFR-1 and VEGFR-2 were constructed and successfully validated in an orthotopic murine tumor model.
authors.affiliation: Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York.
authors.forename: Jan-Philip
authors.initials: JP
authors.lastname: Meyer
authors.affiliation: Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York.
authors.forename: Kimberly J
authors.initials: KJ
authors.lastname: Edwards
authors.affiliation: Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York.
authors.forename: Paul
authors.initials: P
authors.lastname: Kozlowski
authors.affiliation: SibTech Inc., Brookfield, Connecticut.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Backer
authors.affiliation: SibTech Inc., Brookfield, Connecticut lewisj2@mskcc.org jbacker@sibtech.com.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Backer
authors.affiliation: Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York lewisj2@mskcc.org jbacker@sibtech.com.|Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, New York; and.|Weill Cornell Medical College, New York, New York.
authors.forename: Jason S
authors.initials: JS
authors.lastname: Lewis
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D007553:Isotope Labeling; D007554:Isotopes / Q000737:chemistry / Q000493:pharmacokinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D057054:Molecular Imaging / Q000379:methods; D009374:Neoplasms, Experimental / Q000000981:diagnostic imaging* / Q000378:metabolism*; D049268:Positron-Emission Tomography / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000493:pharmacokinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000493:pharmacokinetics*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*; D015040:Zirconium / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: D007554:Isotopes; D019275:Radiopharmaceuticals; D042461:Vascular Endothelial Growth Factor A; D015040:Zirconium; C000592875:Kdr protein, mouse; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: 89Zr PET imaging; VEGFR-1; VEGFR-2; angiogenesis; receptor selectivity