18F Site-Specific Labelling of a Single-Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography

Abstract

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.

18F‐Fluorination of a supported 2‐(aryl‐di‐tert‐butylsilyl)‐N‐methyl‐imidazole for indirect 18F‐labeling of a VHH single‐variable domain

“…Indeed, a non‐coupled PG will accommodate harsher 18 F‐fluorination conditions. Then, the resulting 18 F‐labeled PG can be linked to the antibody fragments/SVD using a mild bioconjugation reaction, such as acylation, 7–13 inverse electron‐demand Diels–Alder reactions (IEDDA), 14–16 thiol‐ene condensation, 17 or strain‐promoted alkyne–azide cycloadditions (SPAACs) 18 …”

“…Indeed, a non-coupled PG will accommodate harsher 18 F-fluorination conditions. Then, the resulting 18 F-labeled PG can be linked to the antibody fragments/ SVD using a mild bioconjugation reaction, such as acylation, [7][8][9][10][11][12][13] inverse electron-demand Diels-Alder reactions (IEDDA), [14][15][16] thiol-ene condensation, 17 or strainpromoted alkyne-azide cycloadditions (SPAACs). 18 In all cases, possible automation of a part or of the whole synthesis remains highly desirable to facilitate the potential transfer to pre-clinical trials.…”

Arginine-Selective Bioconjugation Reagent for Effective 18F-labeling of Native Proteins

“…Alternatively, the proteins are chemically modified or engineered with a functional group, such as hydrazine, (±)-H 3 RESCA, or cysteine. These modified proteins can be radiolabeled with 4-[ 18 F]­fluorobenzaldehyde (4-[ 18 F]­FBA), [ 18 F]­AlF, or N -[2-(4-[ 18 F]­fluorobenzamido)­ethyl]­maleimide (4-[ 18 F]­FBEM), respectively. − However, for clinical use, the modified proteins need to be produced according to the stringent good manufacturing practice (GMP), which adds another significant barrier toward the clinical translation of the protein-based 18 F-PET tracers. Thus, new 18 F-prosthetic groups that can be rapidly radiosynthesized and effectively conjugated with native proteins are highly desirable for the preclinical development of new protein-based PET radiopharmaceuticals and can potentially aid in accelerating their clinical translation.…”