RESUMO
A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortaseâ A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.
Assuntos
Anticorpos Monoclonais/química , Tomografia por Emissão de Pósitrons/métodos , Proteínas Recombinantes/química , Animais , Química Click , Camundongos , Estrutura MolecularRESUMO
Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.
Assuntos
Plaquetas/efeitos dos fármacos , Quelantes/química , Tomografia por Emissão de Pósitrons , Anticorpos de Cadeia Única/química , Animais , Plaquetas/metabolismo , Artérias Carótidas/fisiopatologia , Cobre/química , Radioisótopos de Cobre/química , Diagnóstico por Imagem , Modelos Animais de Doenças , Citometria de Fluxo , Compostos Heterocíclicos com 1 Anel/química , Inflamação , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Ativação Plaquetária , Compostos Radiofarmacêuticos , Trombose/diagnóstico , Microtomografia por Raio-XRESUMO
Site-specific radiolabelling of peptides or antibodies using [(18) F]FBEM is often preferred over non-site-specific radiolabelling with [(18) F]SFB because it does not affect the affinity of the antibody to its target. Unfortunately, the synthesis of [(18) F]FBEM and its conjugation to thiol containing macromolecules requires some manual intervention, which leads to radiation exposure of the radiochemist. In this publication, we report on the complete automation of [(18) F]FBEM production and its subsequent conjugation to glutathione using a slightly modified iPHASE FlexLab module. [(18) F]FBEM was produced in 1.185 ± 0.168 GBq (15-20%; n = 10; 0.75 ± 0.106 GBq non-decay corrected) with a specific activity of 57 ± 10 GBq/µmol. Radiochemical purity was 97 ± 1% and the synthesis time including HPLC purification and reformulation was 70 min. After evaporation to dryness, [(18) F]FBEM was conjugated to glutathione in PBS buffer pH 7.4 in quantitative yields. This fully automated method does not require any manual intervention and therefore reduces the radiation exposure to the operator.
