title: Chemoenzymatic strategy for the synthesis of site-specifically labeled immunoconjugates for multimodal PET and optical imaging.
abstract: The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal (89)Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with (89)Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In in vivo PET and fluorescence imaging experiments, a hybrid (89)Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts.
authors.affiliation: Department of Radiology, Memorial Sloan Kettering Cancer Center , New York, New York 10065, United States.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Zeglis
authors.forename: Charles B
authors.initials: CB
authors.lastname: Davis
authors.forename: Dalya
authors.initials: D
authors.lastname: Abdel-Atti
authors.forename: Sean D
authors.initials: SD
authors.lastname: Carlin
authors.forename: Aimei
authors.initials: A
authors.lastname: Chen
authors.forename: Robert
authors.initials: R
authors.lastname: Aggeler
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Agnew
authors.forename: Jason S
authors.initials: JS
authors.lastname: Lewis
mesh_terms: D000818:Animals; D015179:Colorectal Neoplasms / Q000175:diagnosis; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry; D005690:Galactose / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry* / Q000493:pharmacokinetics; D051379:Mice; D064847:Multimodal Imaging; D009374:Neoplasms, Experimental / Q000175:diagnosis; D061848:Optical Imaging / Q000379:methods*; D011134:Polysaccharides / Q000737:chemistry / Q000276:immunology; D049268:Positron-Emission Tomography / Q000379:methods*; D011868:Radioisotopes; D014018:Tissue Distribution; D023041:Xenograft Model Antitumor Assays; D015040:Zirconium
chemical_list: D005456:Fluorescent Dyes; D018796:Immunoconjugates; D011134:Polysaccharides; D011868:Radioisotopes; D015040:Zirconium; D005690:Galactose