All sorted: The enzyme Sortase A was used to catalyze functionalization of PEGylated capsules with an activation-specific anti-platelet single-chain antibody (scFv). This enzymatic method allows fast, covalent, and site-directed functionalization of delivery vehicles under mild conditions. Activation-specific anti-platelet scFv-coated PEGylated capsules exhibited a high level of selective binding to thrombi, thus suggesting their potential for thrombosis therapy.

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.

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Das Enzym Sortase A zur Funktionalisierung PEGylierter Kapseln mit einem aktivationsspezifischen einkettigen Antikörper (scFv) gegen Plättchen ermöglicht die schnelle, kovalente und ortsspezifische Funktionalisierung von Transportvehikeln unter milden Bedingungen. Mit scFv bedeckte PEGylierte Kapseln binden hoch selektiv an Thrombi, was auf einen möglichen Einsatz in der Thrombosetherapie hindeutet.

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The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically‐replicating negative‐sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA‐damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub‐nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.

Au nique 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 Sortase Af or incorporation of strained cyclooctyne functional groups.T he 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 apositron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets,p latelet aggregates,a nd thrombi, respectively.T his flexible and versatile approach could be readily adapted to incorporate al arge arrayo ft ailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non-site-specific bio conjugation approach with N-succinimidyl-4-[18F]fluorobenzoate (S[18F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine-18 PET radiotracer, based on this antibody, using site-specific bio conjugation to engineer cysteine residues with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). ScFvanti-LIBS and control antibody mut-scFv, with engineered C-terminal cysteine, were reduced, and then, they reacted with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Radiolabelled scFv was injected into mice with FeCl3-induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non-injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer 18F-scFvanti-LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine-18 variant of the scFvanti-LIBS against activated platelets using site-specific bio conjugation.