title: Characterization of 64Cu-DOTA-conatumumab: a PET tracer for in vivo imaging of death receptor 5.
abstract: Conatumumab is a fully human monoclonal antibody that binds to and activates human death receptor 5 (DR5; also known as TRAIL receptor 2). The purpose of this study was to characterize (64)Cu-labeled conatumumab as a PET tracer for imaging DR5 in tumors.
  
  
  METHODS
  DOTA-conatumumab was synthesized by incubating conatumumab with 2,2',2″-(10-(2-(2,5-dioxopyrrolidin-1-yloxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-NHS). The absolute numbers of DOTA molecules per conatumumab molecules were determined by matrix-assisted laser desorption ionization mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry. (64)Cu-DOTA-conatumumab was prepared by incubating (64)CuCl(2) (33-222 MBq) with DOTA-conatumumab at 37°C for 1 h. Binding of conatumumab and DOTA-conatumumab to Fc-coupled human DR5 (huTR2-Fc) was tested in a kinetic analysis assay, and the biologic activity of copper-DOTA-conatumumab was measured using a caspase-3/7 luminescent assay. In vivo evaluation of DOTA-conatumumab and copper-DOTA-conatumumab was done in severe combined immunodeficiency mice bearing Colo205 xenografts: tissue uptake was determined with biodistribution studies, and small-animal PET and autoradiography were used to determine the uptake of (64)Cu-DOTA conatumumab into tumors and other tissues.
  
  
  RESULTS
  DOTA-conatumumab was prepared with an average of 5 DOTA molecules per conatumumab molecule. The in vitro median effective concentration required to induce a 50% effect of DOTA-conatumumab and conatumumab from the assay were 389 and 320 pM, respectively. The median effective dose (±SD) of DOTA-conatumumab and conatumumab via the caspase assay was 135 ± 31 and 128 ± 30 pM, respectively. In female CB17 severe combined immunodeficiency mice bearing Colo205 xenografts, DOTA-conatumumab and conatumumab inhibited tumor growth to the same extent. Small-animal PET studies showed tumor uptake at 24 h after injection of the tracer, with a mean standardized uptake value of 3.16 (n = 2). Tumor uptake was decreased by the coadministration of 400 μg of unlabeled conatumumab (mean standardized uptake value, 1.55; n = 2), suggesting saturable uptake. Tissue uptake determined by biodistribution studies was in agreement with the small-animal PET findings.
  
  
  CONCLUSIONS
  These results suggest that (64)Cu-DOTA-conatumumab is a potential PET tracer for imaging DR5 in tumors and may be useful for measuring on-target occupancy by conatumumab.
authors.affiliation: The Edward Mallinckrodt Institute of Radiology, Washington University, St Louis, Missouri 91320-1799, USA.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Rossin
authors.forename: Tadahiko
authors.initials: T
authors.lastname: Kohno
authors.forename: Aviv
authors.initials: A
authors.lastname: Hagooly
authors.forename: Terry
authors.initials: T
authors.lastname: Sharp
authors.forename: Brian
authors.initials: B
authors.lastname: Gliniak
authors.forename: Thomas
authors.initials: T
authors.lastname: Arroll
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.forename: Art
authors.initials: A
authors.lastname: Hewig
authors.forename: Paula
authors.initials: P
authors.lastname: Kaplan-Lefko
authors.forename: Greg
authors.initials: G
authors.lastname: Friberg
authors.forename: Robert
authors.initials: R
authors.lastname: Radinsky
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Evelhoch
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Welch
authors.forename: Dah-Ren
authors.initials: DR
authors.lastname: Hwang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001345:Autoradiography; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D006207:Half-Life; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D016513:Mice, SCID; D009369:Neoplasms / Q000000981:diagnostic imaging; D009942:Organometallic Compounds / Q000138:chemical synthesis / Q000494:pharmacology; D049268:Positron-Emission Tomography; D019275:Radiopharmaceuticals / Q000138:chemical synthesis / Q000494:pharmacology; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution
chemical_list: C559847:64Cu-DOTA-conatumumab; D000911:Antibodies, Monoclonal; D009942:Organometallic Compounds; D019275:Radiopharmaceuticals; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053148:Caspase 3; D053179:Caspase 7