title: Comparison of DOTA and NODAGA as chelators for (64)Cu-labeled immunoconjugates.
abstract: BACKGROUND
  Bifunctional chelators have been shown to impact the biodistribution of monoclonal antibody (mAb)-based imaging agents. Recently, radiolabeled 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-peptide complexes have demonstrated improved in vivo stability and performance compared to their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) counterparts. Here, we investigated if similar utility could be achieved with mAbs and compared (64)Cu-labeled DOTA and NODAGA-immunoconjugates for the detection of epithelial cell adhesion molecule (EpCAM) in a prostate cancer model.
  
  
  METHODS
  DOTA and NODAGA-immunoconjugates of an EpCAM targeting mAb (mAb7) were synthesized and radiolabeled with (64)Cu (DOTA: 40°C for 1hr; NODAGA: 25°C for 1hr). The average number of chelators per mAb was quantified by isotopic dilution, and the biological activity of the immunoconjugates was evaluated by flow cytometry and ELISA. Radioligand assays were performed to compare cellular uptake and determine the dissociation constant (Kd) and maximum number of binding sites (Bmax) for the immunoconjugates using DsRed-transfected PC3-cells. A PC3-DsRed xenograft tumor model was established in nude mice and used to perform biodistribution studies to compare organ uptake and pharmacokinetics.
  
  
  RESULTS
  (64)Cu-DOTA-mAb7 and (64)Cu-NODAGA-mAb7 were prepared with chelator/protein ratios of 2-3 and obtained in comparable radiochemical yields ranging from 59 to 71%. Similar immunoreactivity was observed with both agents, and mock labeling studies indicated that incubation at room temperature or 40°C did not affect potency. (64)Cu-NODAGA-mAb7 demonstrated higher in vitro cellular uptake while (64)Cu-DOTA-mAb7 had higher Kd and Bmax values. From the biodistribution data, we found similar tumor uptake (13.44±1.21%ID/g and 13.24±4.86%ID/g for (64)Cu-DOTA-mAb7 and (64)Cu-NODAGA-mAb7, respectively) for both agents at 24hr, although normal prostate tissue was significantly lower for (64)Cu-NODAGA-mAb7. (64)Cu-NODAGA-mAb7 also had less accumulation in the liver, suggesting excellent retention of the chelation complex in vivo. This was further confirmed by the higher blood activity of (64)Cu-NODAGA-mAb7, which corresponds to increased bioavailability afforded by the enhanced in vivo stability of the agent. Although tumor/muscle ratios were comparable, tumor/prostate ratios were >2-fold and 1.5-fold higher for (64)Cu-NODAGA-mAb7 at 24 and 48hr, respectively, and suggest better ability to discriminate tumor tissue with (64)Cu-NODAGA-mAb7 in our prostate cancer model.
  
  
  CONCLUSIONS
  To the best of our knowledge, this study represents the first comparison of (64)Cu-labeled DOTA and NODAGA immunoconjugates in vivo. Our results show favorable in vivo performance for (64)Cu-NODAGA-mAb7 which builds upon previous data on our hybrid mAb7 imaging agent by increasing the detection sensitivity for metastatic prostate tumors, as well as for other types of cancer that express EpCAM.
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, USA.
authors.forename: Sukhen C
authors.initials: SC
authors.lastname: Ghosh
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, USA.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Pinkston
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, USA.
authors.forename: Holly
authors.initials: H
authors.lastname: Robinson
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, USA.
authors.forename: Barrett R
authors.initials: BR
authors.lastname: Harvey
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, USA.
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Wilganowski
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Gore
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, USA.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Sevick-Muraca
authors.affiliation: Center for Molecular Imaging, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas Health Science Center at Houston, Houston, TX, USA. Electronic address: ali.azhdarinia@uth.tmc.edu.
authors.forename: Ali
authors.initials: A
authors.lastname: Azhdarinia
mesh_terms: D000085:Acetates / Q000737:chemistry*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001692:Biological Transport; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000737:chemistry*; D003301:Copper Radioisotopes; D004355:Drug Stability; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D008297:Male; D051379:Mice; D014018:Tissue Distribution
chemical_list: C572723:1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane; D000085:Acetates; D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D003301:Copper Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring; D018796:Immunoconjugates; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid
keywords: Antibody; Copper-64; DOTA; EpCAM; NODAGA; PET