Bifunctional sulfur-rich macrocyclic chelators and their immunoconjugates for the targeted delivery of theranostic mercury-197

Abstract

The use of chemically matched theranostic radiometals in nuclear medicine presents a paradigm shift in personalized medicine with immense potential to treat advanced cancers. The nuclear isomers, possess optimal physical decay properties to be applied in theranostic radiopharmaceuticals; however, their use has been limited due to the lack of suitable bifunctional chelators (BFCs) capable of attaching the radionuclides to disease targeting biomolecules. Herein we report the development and evaluation of two novel 197m/gHg BFCs derived from a 15-membered thiacrown ether macrocycle (NS4) bearing isothiocyanate (-NCS) or tetrazine (-Tz) bifunctional handles to allow conjugation to biomolecules. Both chelators were synthesized and radiolabeled with 197m/g Hg, assessed for complex stability, and bioconjugation to trastuzumab (TmAb), a monoclonal antibody targeting HER2 receptors. NS4-Tz efficiently and stably complexes [197m/gHg]Hg2+ and exhibited excellent in vitro stability in both glutathione and human serum. In contrast, NS4-NCS showed lower radiometal incorporation yields and reduced complex stability, likely attributed to non-specific interactions of the isothiocyanate group with Hg2+ . NS4-Tz was successfully conjugated to transcyclooctene-modified TmAb with favourable chelator-to-antibody ratios and subsequently radiolabeled. Due to non-specific Hg2+ binding to TmAb observed during direct labeling, a two-step labeling strategy was employed to improve selectivity. The resulting [197m/gHg]Hg-NS4-Tz-TmAb construct demonstrated specific binding to HER2-positive SK-BR-3 cells in vitro and, in the first in vivo study of a [197m/gHg]Hg-labeled immunoconjugate, confirmed tumour-specific uptake in a SKOV-3 xenograft mouse model. Biodistribution and SPECT/CT studies of the BFC complex alone, [197m/g Hg]Hg-NS4-Tz, revealed high hepatic and splenic accumulation, with some renal uptake possible due to transchelation or tracer pharmacokinetics. While long-term in vivo stability of the radioimmunoconjugate remains a challenge, NS4-Tz shows significant promise for applications with faster-clearing vectors such as peptides or small molecules. Future work will focus on improving hydrophilicity and further optimizing chelator design for mercury-based theranostics.