title: Platinum(II) as bifunctional linker in antibody-drug conjugate formation: coupling of a 4-nitrobenzo-2-oxa-1,3-diazole fluorophore to trastuzumab as a model.
abstract: The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore and trastuzumab (Herceptin™) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L-NBD)Cl](NO3 ) (en=ethylenediamine), with L=N-heteroaromatic, N-alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO4 or Na2 SO4 in tricine or HEPES buffer. Reaction of N-coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5-2.9 that were stable in phosphate-buffered saline at room temperature for at least 48 h. In contrast, thioether-coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.
authors: Department of Radiology and Nuclear Medicine, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam (The Netherlands). d.waalboer@vumc.nl.
authors: Dennis C J
authors: DC
authors: Waalboer
authors: Joey A
authors: JA
authors: Muns
authors: Niels J
authors: NJ
authors: Sijbrandi
authors: Richard B M
authors: RB
authors: Schasfoort
authors: Rob
authors: R
authors: Haselberg
authors: Govert W
authors: GW
authors: Somsen
authors: Hendrik-Jan
authors: HJ
authors: Houthoff
authors: Guus A M S
authors: GA
authors: van Dongen
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D018796:Immunoconjugates / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D009944:Organoplatinum Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D010069:Oxadiazoles / Q000737:chemistry*; D000068878:Trastuzumab / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D018796:Immunoconjugates; D009944:Organoplatinum Compounds; D010069:Oxadiazoles; C011514:4-nitrobenzofurazan; D000068878:Trastuzumab
keywords: antibodies; fluorescent probes; platinum; protein modification; thiourea