title: Synthesis and testing of a binary catalytic system for imaging of signal amplification in vivo.
abstract: We report a binary targeted enzymatic system that is composed of two covalent monoclonal antibody conjugates for specific labeling of cellular targets in vivo. The system utilizes low-molecular weight peroxidase-reducing substrates synthesized by linking 5-hydroxytryptamine (serotonin) with DTPA (5HT-DTPA) for magnetic resonance and radionuclide imaging or with Cy5.5 for near-infrared optical imaging. Initially, the conjugation reaction conditions were optimized to achieve a low level of antiepidermal growth factor receptor (EGFR) antibody (EMD 72000) modification with the N-hydroxysuccinimide ester of 4-hydrazinonicotinate acetone hydrazone (SANH), yielding mAb-HNH conjugate. The resultant modified antibodies were incubated with the periodate-oxidized peroxidase (HRP) or 4-formylbenzoyl-conjugated glucose oxidase (GO), followed by the purification of the resultant mAb-enzyme conjugates by size-exclusion HPLC. The conjugates were further characterized by electrophoresis and were tested by cross-titration on A431 EGFR+ squamous carcinoma or SW620 adenocarcinoma cells (negative control). The conjugates at the optimized concentration ratios were further tested using near-infrared fluorescence microscopy in the presence of Cy5.5 monocarboxy-5-hydroxytryptamide. Further in vitro experiments demonstrated that (1) antibody binding was specific and could be inhibited by free antibody; (2) both antibody conjugates exhibited high enzymatic activity after the binding to the cells; (3) 111In-labeled 5-HT-DTPA was avidly binding to EGFR-positive cells only if both HRP- and GO-conjugates were bound to the cells. The conjugates were tested in vivo using a SPECT imaging experiment, which demonstrated the accumulation of 111In-labeled 5-HT-DTPA substrate at the site containing both conjugates.
authors.affiliation: Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. Alexei.Bogdanov@umassmed.edu
authors.forename: Alexei
authors.initials: A
authors.lastname: Bogdanov
authors.forename: Hye-Won
authors.initials: HW
authors.lastname: Kang
authors.forename: Manuel
authors.initials: M
authors.lastname: Querol
authors.forename: P Hendrik
authors.initials: PH
authors.lastname: Pretorius
authors.forename: Anna
authors.initials: A
authors.lastname: Yudina
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007205:Indium Radioisotopes; D008279:Magnetic Resonance Imaging; D051379:Mice; D008856:Microscopy, Fluorescence; D004369:Pentetic Acid / Q000737:chemistry / Q000378:metabolism*; D012701:Serotonin / Q000737:chemistry / Q000378:metabolism*; D015899:Tomography, Emission-Computed, Single-Photon
chemical_list: D000911:Antibodies, Monoclonal; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007205:Indium Radioisotopes; D012701:Serotonin; D004369:Pentetic Acid; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D066246:ErbB Receptors