RESUMEN
Due to their target specificity, antibody-drug conjugatesâmonoclonal antibodies conjugated to a cytotoxic moietyâare efficient therapeutics that can kill malignant cells overexpressing a target gene. Linking an antibody with radioisotopes (radioimmunoconjugates) enables powerful diagnostics and/or closely related therapeutic applications, depending on the isotope. To generate site-specific radioimmunoconjugates, we utilized genetic code expansion and subsequent conjugation by inverse electron-demand Diels-Alder cycloaddition reactions. We show that, using this approach, site-specific labeling of trastuzumab with either zirconium-89 (89Zr) for diagnostics or lutetium-177 (177Lu) for therapeutics yields efficient radioimmunoconjugates. Positron emission tomography imaging revealed a high accumulation of site-specifically 89Zr-labeled trastuzumab in tumors after 24 h and low accumulation in other organs. The corresponding 177Lu-trastuzumab radioimmunoconjugates were comparably distributed in vivo.
Asunto(s)
Inmunoconjugados , Radioisótopos , Tomografía de Emisión de Positrones/métodos , Anticuerpos Monoclonales , Trastuzumab , Línea Celular Tumoral , Marcaje Isotópico/métodosRESUMEN
Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels-Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to ß-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a)â the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo ß-elimination and b)â a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.
Asunto(s)
Alquinos , Aminoácidos , Animales , Reacción de Cicloadición , Colorantes Fluorescentes , ProteínasRESUMEN
We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.
Asunto(s)
Proteínas Fluorescentes Verdes/biosíntesis , Complejos Multiproteicos/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Virales/biosíntesis , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Técnicas de Cultivo de Célula , Transferencia Resonante de Energía de Fluorescencia/métodos , Código Genético , Vectores Genéticos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The stereoselective synthesis of a variety of functionalized hydroindoles suitable as building blocks for thiodiketopiperazine natural products such as rostratins B-D is reported. The key precursor for all transformations is a previously reported hexahydroindole compound. All functional groups were installed with the desired stereochemistry and the feasibility of the synthetic strategy was exemplified by dimerization of two hydroindole units to form the pentacyclic C2 -symmetric scaffold of rostratinâ C.