title: Bioorthogonal Fluorophore Linked DFO-Technology Enabling Facile Chelator Quantification and Multimodal Imaging of Antibodies.
abstract: Herein we describe the development and application of a bioorthogonal fluorogenic chelate linker that can be used for facile creation of labeled imaging agents. The chelate linker is based on the trans-cyclooctene(TCO)-tetrazine(Tz) chemistry platform and incorporates deferoxamine (DFO) as a (89)Zr PET tracer and a BODIPY fluorophore for multimodal imaging. The rapid (<3 min) ligation between mAb-TCO and Tz-BODIPY-DFO chelator is monitored using fluorescence and allows for determination of labeling completion. Utilizing BODIPY as the linker between mAb and DFO facilitates in chelator quantification using spectrophotometry, allowing for an alternative to traditional methods (mass and isotope dilution assay). Radiolabeling with (89)Zr to form (89)Zr-DFO-BODIPY-trastuzumab was found to be quantitative after incubation at room temperature for 1 h (1.5 mCi/mg specific activity). The cell binding assay using HER2+ (BT474) and HER2- (BT20) cell lines showed significant binding to (89)Zr-DFO-BODIPY-trastuzumab (6.45 ± 1.87% in BT474 versus 1.47 ± 0.39% in BT20). In vivo PET imaging of mice bearing BT20 or BT474 xenografts with (89)Zr-DFO-BODIPY-trastuzumab showed high tumor conspicuity, and biodistribution confirmed excellent, specific probe uptake of 237.3 ± 14.5% ID/g in BT474 xenografts compared to low, nonspecific probe uptake in BT20 xenografts (16.4 ± 5.6% ID/g) 96 h p.i. . Ex vivo fluorescence (465ex/520em) of selected tissues confirmed superb target localization and persistence of the fluorescence of (89)Zr-DFO-BODIPY-trastuzumab. The described platform is universally adaptable for simple antibody labeling.
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital , 185 Cambridge Street, CPZN 5206, Boston, Massachusetts 02114, United States.
authors.forename: Labros G
authors.initials: LG
authors.lastname: Meimetis
authors.affiliation: The Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital , 149 Thirteenth street, Charlestown, Massachusetts 02129, United States.
authors.forename: Eszter
authors.initials: E
authors.lastname: Boros
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital , 185 Cambridge Street, CPZN 5206, Boston, Massachusetts 02114, United States.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Carlson
authors.affiliation: The Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital , 149 Thirteenth street, Charlestown, Massachusetts 02129, United States.
authors.forename: Chongzhao
authors.initials: C
authors.lastname: Ran
authors.affiliation: The Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital , 149 Thirteenth street, Charlestown, Massachusetts 02129, United States.
authors.forename: Peter
authors.initials: P
authors.lastname: Caravan
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital , 185 Cambridge Street, CPZN 5206, Boston, Massachusetts 02114, United States.|Department of Systems Biology, Harvard Medical School , 200 Longwood Avenue, Boston, Massachusetts 02115, United States.
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001896:Boron Compounds / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry*; D003676:Deferoxamine / Q000737:chemistry; D005260:Female; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics; D006801:Humans; D008819:Mice, Nude; D064847:Multimodal Imaging / Q000379:methods*; D049268:Positron-Emission Tomography / Q000379:methods; D011868:Radioisotopes / Q000737:chemistry; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D014018:Tissue Distribution; D000068878:Trastuzumab; D023041:Xenograft Model Antitumor Assays; D015040:Zirconium / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D001896:Boron Compounds; D002614:Chelating Agents; D003676:Deferoxamine; D005456:Fluorescent Dyes; D011868:Radioisotopes; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; D015040:Zirconium; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; C508053:ERBB2 protein, human