MANOTA: a promising bifunctional chelating agent for copper-64 immunoPET

Abstract

Improved bifunctional chelating agents (BFC) are required for copper-64 radiolabelling of monoclonal antibodies (mAbs) under mild conditions to yield stable, target-specific imaging agents. Four different bifunctional chelating agents (BFC) were evaluated for Fab (Fragment antigen binding) conjugation and radiolabelling with copper-64. Two DOTA- (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and two NOTA- (1,4,7-triazacyclononane-1,4,7-triacetic acid) derivatives bearing a p-benzyl-isothiocyanate group were conjugated to Fab-trastuzumab - which targets the HER2/neu receptor - and the average number of chelators attached ranged from 2.4 to 4.3 macrocycles per Fab. Labelling of the immunoconjugate with copper-64 was achieved in high radiochemical yields after 45 min at 37 °C, and the radiochemical purity of each Cu-BFC-Fab-trastuzumab reached 97% after purification. The affinity of eachCu-BFC-Fab-trastuzumab ranged between 10 and 50 nM as evaluated by in vitro saturation assays using the HCC1954 breast cancer cell line. PET-MR imaging and biodistribution studies were performed in mice bearing breast cancer BT-474 xenografts. BT-474 tumours were clearly visualized on PET images at 4 and 24 hours post-injection. The tumour uptake of Cu-BFC-Fab-trastuzumab reached 8.9 to 12.8% ID g 24 hours post-injection and significant differences in non-specific liver uptake were observed depending on the BFC conjugated, the lowest being observed with MANOTA. These results show that MANOTA is a valuable tool for copper-64 radiolabelling.

Methylthiazolyl Tacn Ligands for Copper Complexation and Their Bifunctional Chelating Agent Derivatives for Bioconjugation and Copper-64 Radiolabeling: An Example with Bombesin

“…The results obtained with [ 64/67 Cu­( nota )] − radiopharmaceuticals were quite encouraging, especially compared with the H 4 dota analogue, which is now recognized in nuclear medicine as an inappropriate chelator for both copper­(II) isotopes because of the low kinetic inertness of the corresponding complexes . On the other hand, H 3 nota was shown to be a promising 64 Cu chelator (few examples with 67 Cu are known considering its currently limited availability) and used in various bifunctional forms through its conjugation with biovectors . However, it is now admitted that, even if no one could predict it, the influence of the metal complexes on the biological properties is not linked only to the coordination properties of the chelates.…”

“…5 On the other hand, H 3 nota was shown to be a promising 64 Cu chelator (few examples with 67 Cu are known considering its currently limited availability) and used in various bifunctional forms through its conjugation with biovectors. 6 However, it is now admitted that, even if no one could predict it, the influence of the metal complexes on the biological properties is not linked only to the coordination properties of the chelates. Indeed, depending on the biological targets, it is important to have an array of complexes of different size, shape, lipophilicity, and charge.…”

The Chemical Scaffold of Theranostic Radiopharmaceuticals: Radionuclide, Bifunctional Chelator, and Pharmacokinetics Modifying Linker

“…The 64 Cu-MANOTA complex was not transchelated in the presence of EDTA, indicating high stability of the complex. In vivo stability of 64 Cu-MANOTA immunoconjugate was demonstrated by low uptake from non-target organs and a high ratio of 64 Cu-MANOTA in target organs [ 187 ].…”

Monoclonal antibody-based molecular imaging strategies and theranostic opportunities

“…Their results demonstrated that the chelator MANOTA (Methyl Amino triazacycloNOnane Triacetic Acid) was the most efficient. Their conjugation strategy was based on an isothiocyanate group and involved linkage to the free amine on the lysine residues 30.…”