Radiolabelling small and biomolecules for tracking and monitoring

Abstract

Radiolabelling small molecules with beta-emitters has been intensively explored in the last decades and novel concepts for the introduction of radionuclides continue to be reported regularly. New catalysts that induce carbon/hydrogen activation are able to incorporate isotopes such as deuterium or tritium into small molecules. However, these established labelling approaches have limited applicability for nucleic acid-based drugs, therapeutic antibodies, or peptides, which are typical of the molecules now being investigated as novel therapeutic modalities. These target molecules are usually larger (significantly >1 kDa), mostly multiply charged, and often poorly soluble in organic solvents. However, in preclinical research they often require radiolabelling in order to track and monitor drug candidates in metabolism, biotransformation, or pharmacokinetic studies. Currently, the most established approach to introduce a tritium atom into an oligonucleotide is based on a multistep synthesis, which leads to a low specific activity with a high level of waste and high costs. The most common way of tritiating peptides is using appropriate precursors. The conjugation of a radiolabelled prosthetic compound to a functional group within a protein sequence is a commonly applied way to introduce a radionuclide or a fluorescent tag into large molecules. This review highlights the state-of-the-art in different radiolabelling approaches for oligonucleotides, peptides, and proteins, as well as a critical assessment of the impact of the label on the properties of the modified molecules. Furthermore, applications of radiolabelled antibodies in biodistribution studies of immune complexes and imaging of brain targets are reported.

Fig. 1. Schematic sketches of (1) bispecific antibody based on knobs-into-holes-technology; (2) antibody conjugates. The red star symbolises payloads such as a small molecule, peptide, or oligonucleotide; (3) peptide; (4) antisense oligonucleotide in green with mRNA in red.

Fig. 2. Schematic reaction illustration of a radionuclide complexing bifunctional chelator linked to a carrier molecule, e.g. oligonucleotide, peptide, antibody.

Fig. 3. Literature reported positions for radiolabelling of oligonucleotides in example of thymidine and adenosine. Colouring reflects the corresponding radioisotopes. Blue: ³H; green: ¹⁴C; yellow: ³⁵S.

Fig. 4. General synthetic route to ASOs containing tritium labelled propionate functionalisation on C₆-amine linker.

Fig. 5. Synthesis routes to tritium-labelled maleimide derivative. (A) Metal-catalytic C–H activation followed by hydrogen/tritium exchange using tritium gas. (B) Photoredox-mediated hydrogen/tritium exchange using tritiated water. (C) Reduction of double/triple-bonds. (D) Palladium-catalysed halogen/tritium exchange. (E) Methylation on nucleophilic residue using [³H]-methyl nosylate. Yellow arrows indicate possible labelling positions.

Fig. 6. Current synthetic routes to tritium-labelled peptides.

Fig. 7. Quality control has to be integrated into a typical labelling process for mAbs used in biological studies.

Fig. 8. Neutralising and non-neutralising drug-ADA immune complexes.

Fig. 9. Chemical structures of irreversible and reversible MAGL-PET tracers.

Fig. 10. Inverse electron-demand Diels–Alder ligation between a TCO linked to a carrier moitey and a tagged Tz derivative.

Fig. 11. Reaction kinetics from slow (left) to high (right) of selected Tzs with TCO in PBS at 37 °C and corresponding stability assessed in PBS at 37 °C for 10 h.

Fig. 12. Examples of strained dienophiles for IEDDA reactions, from left to right with slow to high reaction kinetics.

Fig. 13. Pretargeting workflow. In the first step, a BBB-penetrating TCO-modified antibody is injected. The mAb is actively transported across the BBB and binds to a target. In the second step, a PET nuclide-labelled Tz is injected and clicks with the TCO-mAb to generate PET-images of misfolded proteins.

Martin R. Edelmann