Labelling of Small and Biomolecules for Tracking and Monitoring

: (Alternative Format Thesis)

Abstract

This thesis opens with a critical review of the literature on radiolabelling small and biomolecules for tracking and monitoring. A novel concept of tritium labelling of antisense oligonucleotides via different conjugation agents was developed. It was demonstrated in in vitro and in vivo experiments that the conjugation of [³H]N-ethylmaleimide (NEM) to a thiol linker placed on an oligonucleotide has no impact on the pharmacokinetic (PK) behaviour compared to the unlabelled drug. A robust synthetic route was developed for a tritium-O-methylation of N-alkoxy maleimide derivatives that can be used as labelling reagents for biomolecules. In a 3-step synthesis, tritium-labelled maleimide derivatives were efficiently prepared that are significantly less volatile than NEM and therefore safer to handle in the laboratory with respect to radiation protection. Selective tritium labelling of neuromedin S (NMS) by derivatisation with [³H]N-succinimidylpropionate (NSP) was established. A functional ligand binding assay showed that labelling with NSP leads to a comparable result to unlabelled NMS, regardless of the degree of labelling and labelling position. With respect to antibody labelling, a functional in vitro assessment workflow for modified (fluorescently labelled or radiolabelled) antibodies was developed to predict the possible impact of labelling to protein properties or PK behaviour. Tritium-labelled therapeutic antibody was used in a biodistribution study of drug/anti-drug antibody (ADA) immune complexes (IC) to investigate the impact of IC formation on drug distribution. A high level of IC was found in the pancreas in female Wistar rats. Using multi-parameter optimisation, a morpholin-3-one derivative with an improved kinetic binding profile for imaging monoacylglycerol lipase (MAGL) in the central nervous system (CNS) was developed and successfully labelled with ¹¹C. The synthesis of eleven tetrazine (Tz) derivatives for CNS targeted imaging was described. Tzs were evaluated and classified for their properties as potential CNS radiotracers and click partners for bioorthogonal PET imaging based on several in vitro and in vivo experiments. These studies were predominantly performed with non-radioactive Tzs, but also included an in vivo bio-distribution study with fluorine-18 labelled Tzs. Out of the eleven synthesised Tzs, two (Tz 1: 3-(4-methoxyphenyl)-6-methyl-1,2,4,5-tetrazine; Tz 2: N-[2-(2-fluoroethoxy)ethyl]-4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzamide) were proposed for labelling as the most promising candidates that showed the best properties in terms of brain penetration and stability. Tz 1 was successfully tritium labelled and investigated for antibody-based in vivo CNS imaging. Although evaluation of a pretargeting approach using a TCO-conjugated BrainShuttle antibody and tritium-labelled Tz showed an ex vivo click response, in vivo conjugation has been unsuccessful. The isotopes ³H, ¹¹C, and ¹⁸F have been incorporated in biologically relevant ligands for tracking and monitoring.

Date of Award	29 Mar 2023
Original language	English
Awarding Institution	University of Bath
Supervisor	Ian Blagbrough (Supervisor) & Stephen Husbands (Supervisor)