Synthesis and In Vitro Comparison of DOTA, NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates

The development of Cu-64-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [Cu-64]Cu-15-5 complex prompted us to investigate its potential for the Cu-64-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with Cu-64, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [Cu-64]Cu-15-5- and [Cu-64]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88-94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition.

Automated summary

The development of Cu-64-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators for stable copper complexes. In this study, the potential of the pentaazamacrocyclic [Cu-64]Cu-15-5 complex for Cu-64-labelling of monoclonal antibodies (mAbs) was investigated. Different BFCs were synthesized and covalent bonds were formed between them and azide-functionalized trastuzumab using strain-promoted azide-alkyne cycloaddition. The NODAGA- and 15-5-mAb conjugates showed excellent radiolabeling yields with Cu-64, and presented higher resistance to transchelation when challenged by EDTA compared to the DOTA derivative.